CN106434246A - Use of xylanase - Google Patents
Use of xylanase Download PDFInfo
- Publication number
- CN106434246A CN106434246A CN201610802626.3A CN201610802626A CN106434246A CN 106434246 A CN106434246 A CN 106434246A CN 201610802626 A CN201610802626 A CN 201610802626A CN 106434246 A CN106434246 A CN 106434246A
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- seq
- xylanase
- enzyme
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- activity
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
- C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
- C12H1/12—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages without precipitation
- C12H1/14—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages without precipitation with non-precipitating compounds, e.g. sulfiting; Sequestration, e.g. with chelate-producing compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C11/00—Fermentation processes for beer
- C12C11/003—Fermentation of beerwort
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C12/00—Processes specially adapted for making special kinds of beer
- C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
-
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
- C12N9/242—Fungal source
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
- D21H17/005—Microorganisms or enzymes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
The present invention relates to a composition including enzymes having endo-1,4-beta-xylanase activity and enzymes having endo-1,3(4)-beta-glucanase activity. The present invention specifically relates to new enzymes having endo-1,4-beta-xylanase activity and enzymes having endo-1,3(4)-beta-glucanase activity, with improved properties and to compositions comprising these enzymes suitable for use in the production of a food, feed, or malt beverage product, such as in a brewing process.
Description
The application be the applying date be September 14 mesh in 2012 Chinese patent application 201280044661.3 " in comprising to have
Cut-Isosorbide-5-Nitrae-beta-xylanase activity enzyme and there are inscribe -1, the compositionss of the enzyme of 3 (4)-beta glucanase activities " division Shen
Please.
Technical field
The present invention relates to have the enzyme of improved property and be related to comprise be applied to produce for example brewage process food,
The compositionss of these enzymes of beverage (such as medicated beer), feedstuff or bio-fuel.
Background technology
Beer Production is well-known using enzyme.Describe for enzyme to be applied to converted mash system in WO 97/42302
Standby (mashing) step is to improve converted mash (mash) filterability and to improve extractum yield.
WO2005118769 and WO2005059084 be related in process for beer production converted mash preparation and filtration step and
Relate to the enzymatic compositions of this technique.
WO1999057325 is related to the bacterial strain of penicillium funiculosum (Penicillium funiculosum), is related to from wherein
The new enzymatic mixture obtaining and the nucleotide sequence of described enzyme.
However it is still necessary to can be used for improved enzyme and the enzyme combination of the production of Food ＆ Drink product, such as ethanol drink
In converted mash preparation in the production of material such as medicated beer and whiskey, steaming and decocting and filtration step.
The purpose of the present invention
The purpose of embodiment of the present invention is to provide the enzyme of the production being suitable for Food ＆ Drink product, such as in ethanol drink
In the production of material or non-alcoholic beverage (beverage for example based on corn or Fructus Hordei Germinatus such as medicated beer or whiskey).There is provided
Enzyme can have the improved property related to the use in brewageing.These broad category of improved properties include for example optimal
Temperature improve, the activity to solubility araboxylan (WE-AX) substrate with to insoluble araboxylan (WU-AX) bottom
Gross pressure in the ratio improvement of the activity of thing, the filtering (lautering) in brewage process and/or filtration (filtration) step
Raise minimizing and the filterability of enzyme treated material improves.
Content of the invention
It has been found by the present inventors that one or more enzymes and the combination of some enzymes have with respect to known enzyme and enzyme combination
Improved property, described improved property especially using related, used in brewage process and is somebody's turn to do in such brewage process
The starch-containing material of one or more ferment treatment uses converted mash to produce to brewage.
Therefore, in a first aspect, the present invention relates to the enzyme of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity, this enzyme comprise with
Selected from SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、
SEQ ID NO：17 and SEQ ID NO：18 any one or their any function fragment have at least 80% homogeneity
Aminoacid sequence.
" function fragment " used herein refers to the truncate version of enzyme, and its reference enzyme with non-truncated has substantially the same
The enzymatic activity of enzymatic activity or at least significance degree.
Therefore, in second aspect, the present invention relates to display inscribe -1, the enzyme of 3 (4) -1,4 beta-glucanase activities, this enzyme comprises
With selected from SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10 and SEQ ID NO：11、SEQ ID
NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：The appointing of 16 any one or they
What function fragment has the aminoacid sequence of at least 80% homogeneity.
In the third aspect, the present invention relates to a kind of comprise to encode the DNA construct of the DNA sequence of the enzyme according to the present invention.
It yet still another aspect, the present invention relates to a kind of recombinant expression carrier, it includes and comprises to encode the enzyme according to the present invention
DNA sequence DNA construct.
It yet still another aspect, the present invention relates to a kind of cell, it is with comprising to encode the DNA sequence of the enzyme according to the present invention
DNA construct converts.
It yet still another aspect, the present invention relates to comprising the enzyme according to the present invention or DNA construct or carrier or cell
Preparation.
It yet still another aspect, the present invention relates to comprising the enzyme of the display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity according to the present invention
The compositionss combining with any or multiple 1,4 beta-glucanases.
It yet still another aspect, the present invention relates to comprising display inscribe -1 according to the present invention, 3 (4) -1,4 beta-glucanase activities
The compositionss combining of enzyme and any or multiple xylanase.
It yet still another aspect, the present invention relates to the enzyme according to the present invention or the preparation according to the present invention or according to the present invention
Compositionss producing food, the purposes in feedstuff or malt beverage product such as medicated beer and whiskey.
It yet still another aspect, the present invention relates to the enzyme according to the present invention or the preparation according to the present invention or according to the present invention
Purposes in producing dough or the roasting product of training for the compositionss.
It yet still another aspect, the present invention relates to the enzyme according to the present invention or the preparation according to the present invention or according to the present invention
Purposes in preparing paper pulp or paper for the compositionss.
It yet still another aspect, the present invention relates to the enzyme according to the present invention or the preparation according to the present invention or according to the present invention
Compositionss be used for preparing the purposes of grain component.In some embodiments, corn is rye (Secale cereale L.), Semen Tritici aestivi or Fructus Hordei Vulgaris.
It yet still another aspect, the present invention relates to the enzyme according to the present invention or the preparation according to the present invention or according to the present invention
Compositionss producing medicated beer or the purposes during brewage process by-product is modified.
It yet still another aspect, the present invention relates to the enzyme according to the present invention or the preparation according to the present invention or according to the present invention
Compositionss produce wine (wine) or fruit juice in purposes.
It yet still another aspect, the present invention relates to the enzyme according to the present invention or the preparation according to the present invention or according to the present invention
Purposes in producing the first generation or second filial generation bio-fuel such as bio-ethanol for the compositionss.
It yet still another aspect, the present invention relates to a kind of method of the filterability changing starch-containing material, methods described includes
Step with starch-containing material described in the enzyme according to the present invention or preparation or compositions-treated.
It yet still another aspect, the present invention relates to a kind of in brewageing application reduce filtering during pressure rise method, institute
The method of stating includes brewageing the step with converted mash with the enzyme according to the present invention or preparation or compositions-treated.
It yet still another aspect, the present invention relates to one kind be used for producing food, feedstuff or beverage products such as alcoholic beverage or
The method of the non-alcoholic beverage beverage for example based on corn or Fructus Hordei Germinatus such as medicated beer or whiskey, methods described includes using basis
The enzyme of the present invention or the step of preparation or the starch-containing material of compositions-treated.
It yet still another aspect, the present invention relates to a kind of for producing the method with converted mash brewageed, methods described includes using root
According to the enzyme of the present invention or the step of preparation or the starch-containing material of compositions-treated.
It yet still another aspect, the present invention relates to a kind of for producing the first generation or the second filial generation bio-fuel such as bio-ethanol
Method, methods described includes the step with the enzyme according to the present invention or preparation or the starch-containing material of compositions-treated.
It yet still another aspect, a kind of the present invention relates to product being obtained by the method according to the invention.
It yet still another aspect, the present invention relates to a kind of compositionss of the product comprising to obtain by the method according to the invention,
Product is in the range of 0.1%-99.9% as described therein.
Brief description
Fig. 1：Laboratory scale and pilot-scale brewage used in converted mash preparation process (profile).By 10 points
Batch mixing (mashing-in) time of clock starts converted mash preparation, then adds enzyme.
Fig. 2：The pilot-scale deriving from the checking to glucanase and xylanase screening brewages application result.Contrast is blank
WithMax tests the group of bacillus subtilises (B.sub) glucanase S and Tabin aspergillus (A.tub) xylanase
Close.Collected data is that the gross pressure during mean flow rate (L/h), filtering raises (mm WC, wherein 1mm WC=
The maximum pressure (mm WC) recording 9.80665Pa) and during filtering.
Fig. 3：Feature in brewageing for the xylanase
Fig. 4：Apply the flow velocity-filtering of various xylanase material standed fors
Fig. 5：The meansigma methodss of beer filtration-multiple times of filtration
Fig. 6：The meansigma methodss of beer filtration-multiple times of filtration
Fig. 7：The converted mash preparation diagram of embodiment 3.
Specific embodiment
Medicated beer traditionally refers to spread out from Fructus Hordei Germinatus (Fructus Hordei Germinatus such as spreading out from barley grain) and optional adjuvant (as starch-containing plant
Thing material (such as grain grain)) and optional seasoning (for example using Flos lupuli (Flos Humuli Lupuli) seasoning) alcoholic beverage.
In the context of the present invention, term " medicated beer " is intended to including any fermentation/wine by starch yielding plant material
Make the fermenting wort of production, therefore specifically also include specially producing or any combinations from Fructus Hordei Germinatus and adjuvant from adjuvant
The medicated beer producing.
Term " fermentation " means to produce certain material by growth microorganism in culture in the context of the present invention
As ethanol.Generally, fermented using microorganism such as yeast.
Term " Fructus Hordei Germinatus " used herein is understood to the grain kernels of any germination, the Fructus Hordei Vulgaris such as germinateing." adjuvant " can
It is defined as the starch yielding plant material of any non-Fructus Hordei Germinatus or barley malt.
" starch yielding plant material " may be, for example, one or more corn, such as Fructus Hordei Vulgaris, Semen Tritici aestivi, Semen Maydiss, rye (Secale cereale L.), height
Fine strain of millet, millet or rice and any combination of them.Starch yielding plant material can be processed, for example, grind, germinate, part is sent out
Bud or do not germinate.Ablastous corn is also referred to as " raw grain ".The example of non-corn starch yielding plant material includes for example that tuber is such as
Rhizoma Solani tuber osi and Maninot esculenta crantz..
Term " beverage " used herein and " beverage products " include the fermented beverage that such as following meeting forms foam：Entirely
Malt beer (full malted beer), with "《German beer purifying method》" (" Reinheitsgebot ") brewage medicated beer,
High wine degree medicated beer, dry beer, low wine degree medicated beer, light beer, low alcohol beer, low-calorie medicated beer, baud dark beer, Bock beer
Wine, Si Taote dark beer, malt liquor, alcohol-free beer, no alcohol malt liquor etc..Term " beverage " or " beverage products " are also included no
Bubble medicated beer and atypical Fructus Hordei Germinatus beverage, the such as Fructus Hordei Germinatus beverage of fruit flavoured, (the such as Fructus Citri Limoniae seasoning of such as citrus fruit seasoning
, Fructus Citri junoriss seasoning, Citrus aurantium Linn. seasoning or berry seasoning) Fructus Hordei Germinatus beverage, wine seasoning Fructus Hordei Germinatus beverage (for example Little water. adjust
Taste, rum seasoning or mescal seasoning malt liquor), or Fructus Hordei Germinatus beverage (the such as caffeine local flavor of coffee seasoning
Malt liquor) etc..
Medicated beer can be by substantially the same technique from multiple starch yielding plant material manufactures, and wherein starch is mainly by Fructus Vitis viniferae
Sugared homopolymer composition, with α-Isosorbide-5-Nitrae-or α -1,6- is bonded for wherein glucose residue, mostly former key.
The technique manufacturing fermented beverage such as medicated beer is commonly referred to brewageed.Manufacturing traditional raw material that these beverages are used is
Water, Flos lupuli (Flos Humuli Lupuli) and Fructus Hordei Germinatus.Replacement in addition to Fructus Hordei Germinatus or as Fructus Hordei Germinatus, such as following adjuvant can be used as coming of starch
Source：Common corn grits, fine grinding Semen Maydis powder, medicated beer processed are with grinding yeast, rice, Sorghum vulgare Pers., refined corn starch, Fructus Hordei Vulgaris, greatly
Wheat starch, pot barley, Semen Tritici aestivi, wheaten starch, the corn baked and banked up with earth, cereal piece, rye (Secale cereale L.), Herba bromi japonici, Rhizoma Solani tuber osi, Maninot esculenta crantz. and syrup
(as corn syrup, sugarcane syrup, Nulomoline syrup, Fructus Hordei Vulgaris and/or wheat syrup) etc..Starch is finally enzymatically converted to
Fermentable sugars.
For mainly from the medicated beer that Fructus Hordei Germinatus (for example adjuvant is at most 15-20%) manufacture, due to many reasons, Fructus Hordei Germinatus
(it mainly produces from selected barley variety) has maximum impact to the general characteristic of medicated beer and quality.First, Fructus Hordei Germinatus are
Main flavoring agent in medicated beer.Second, Fructus Hordei Germinatus provide the major part of fermentable sugars.3rd, Fructus Hordei Germinatus provide protein, and it is by tribute
Offer wine body and the foam characteristics of medicated beer.4th, Fructus Hordei Germinatus provide necessary enzymatic activity during converted mash preparation.
Also the quality to medicated beer (inclusion local flavor) has contributed much Flos lupuli (Flos Humuli Lupuli).Specifically, Flos lupuli (Flos Humuli Lupuli) (or Flos lupuli (Flos Humuli Lupuli) become
Point) add, in medicated beer, the bitterness compound matter wanted.Additionally, Flos lupuli (Flos Humuli Lupuli) can play protein precipitant effect, set up preservative
And help formation of foam and stabilisation.Not all of medicated beer is all produced using Flos lupuli (Flos Humuli Lupuli).Also can using other stabilizers such as
Protease (such as papain).
Conventional brewage process can be described as follows, but does not want to allow it be interpreted limitation of the present invention：
The technique manufacturing medicated beer is known in the art, but in brief, it is related to five steps：(a) converted mash system
Standby and/or adjuvant steaming and decocting (b) beerwort separates and extracts (c) wort boiling and adds Flos lupuli (Flos Humuli Lupuli) (d) cooling, fermentation and store up
Deposit, and (e) maturation, processing and packaging.Generally, in the first step, will grind or the Fructus Hordei Germinatus of crushing are mixed with water and be subject to
A period of time is kept to allow enzyme present in Fructus Hordei Germinatus by Starch Conversion present in Fructus Hordei Germinatus as fermentable sugars at a temperature of control.
In second step, converted mash is transferred to " filter tank " or converted mash filter, liquid is remaining with grain wherein
Thing separates.This sweet smell liquid is referred to as " beerwort ", and remaining grain residue is referred to as " wheat grain ".Generally converted mash is carried
Take, this is related to add water to converted mash to reclaim remaining solubility extractum from wheat grain.
In the third step, beerwort is acutely boiled.This beerwort is sterilized and contribute to developing colourity, local flavor gentle
Taste.Sometime addition Flos lupuli (Flos Humuli Lupuli) in boiling part.
In the 4th step, beerwort is cooled down and is transferred to fermentation tank, described fermentation tank equipped with yeast or is added thereto to
Yeast.Yeast converts the sugars into alcohol and carbon dioxide by fermentation；Last in fermentation, so that fermentation tank is turned cold or can make to send out
Fermentation tank turns cold to stop fermenting.Remove Yeast Flocculation thing.
In the final step, medicated beer is cooled down and store a period of time, in the described time period, medicated beer becomes clarification and its wind
Taste develops, and any material that may damage beer colours of wine, local flavor and shelf life is all precipitated out.Before encapsulation, right
Medicated beer filling CO 2 gas is simultaneously optionally filtered and pasteurization.
The beverage of the alcohol usually containing about 2 weight % to about 10 weight % after fermentation, can be obtained.Not fermentable carbon water
Compound is not converted during the fermentation and forms the major part of the solid content being dissolved in final medicated beer.
The retaining of this residue be because malto-amylase can not amylatic α -1,6- key.Not fermentable carbon
Hydrate contributes about 50 calories/12 ounces medicated beer.
Recently, the brewed beverages of referred to as table beer, the medicated beer of calorie minimizing or low-calorie medicated beer are widely spread, special
It is not in American market.As defined in the U.S., the calorie of these medicated beer is lower about than " normal " medicated beer of manufacturer
30%.
Used in the technology and brewing technology of the more information with regard to conventional brewing processes and the present invention to be applied to
The definition of term, can find in the following documents：Institute (Research and Teaching Institute of is brewageed in Berlin
Brewing, Berlin (VLB)) Wolfgang Kunze written " Technology Brewing and Malting (《Make
Make and wheat technology processed》) ", second edition revised edition 1999, ISBN 3-921690-39-0, the 3rd edition (2004)：ISBN 3-
921690-49-8, the 4th edition latest edition 2010 (ISBN 978-3-921690-64-2).
Xylanase is classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC 3.2.1.156；It
Activity can for example measure as described in embodiment.Will be with display inscribe -1 according to the present invention, 3 (4)-β-Portugal gathers
The suitable xylanase that the enzyme of anase activity is applied in combination includes classifying in EC 3.2.1.8, EC 3.2.1.32, EC
3.2.1.136 any xylanase with EC 3.2.1.156, such as WO 2010072226, WO 2010072225, WO
2010072224、WO 2005059084、WO2007056321、WO2008023060A、WO9421785、WO2006114095、
Any one disclosed in WO2006066582, US 2008233175 and WO10059424.
Inscribe-Isosorbide-5-Nitrae-β xylanase is classified as EC 3.2.1.8.This enzyme causes the Isosorbide-5-Nitrae-β-D- xyloside in xylan
Interior cut hydrolysis (endohydrolysis) of key.
Term " family 11 xylanase " used herein, " glycoside hydrolase (GH) family 11 " or simply " GH 11
Xylanase " refers to be categorized as the inscribe-Isosorbide-5-Nitrae-β xylanase of EC 3.2.1.8, and it causes the Isosorbide-5-Nitrae-β-D- xylose in xylan
The interior cut hydrolysis of glycosidic bond, and it is classified as family 11 xylanase according to documents below：B.Henrissat, A
classification of glycosyl hydrolases based on amino acid sequence
Similarities (is classified to glycosyl hydrolase based on amino acid sequence similarity), Biochem.J. (《Biochemistry is miscellaneous
Will》), 280 (1991), the 309-316 page.
Term " family 10 xylanase ", " glycoside hydrolase (GH) family 10 " or simply " GH 10 xylanase "
Including the enzyme with multiple known activity, such as xylanase (EC：3.2.1.8)；Inscribe -1,3- beta-xylanase (EC：
3.2.1.32)；Cellobiohydrolase (EC：3.2.1.91).These enzymes are formerly referred to as cellulase family F.
In some embodiments, the enzyme of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity is family 11 xylanase.One
In a little embodiments, Mei Shi family 10 xylanase of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity.
In one aspect, had as measured by by the algoscopy described in embodiment according to the enzymatic compositions of the present invention
Inscribe-Isosorbide-5-Nitrae-β xylanase activity.
The algoscopy of measurement xylanase activity can use xylan as substrate at pH 3.5 or pH 5 and 50 DEG C
Carry out, or it can be carried out to carry out other sign and explanation to enzyme under different pH and Temperature numerical.Often single from xylose
At the 540nm that the position time causes, absorbance incrementss calculate enzymatic activity.
In some embodiments, as measured by by the algoscopy described in embodiment, according to the enzyme combination of the present invention
Thing comprises at least about 5000U/g, such as at least about 6000U/g, such as at least about 7000U/g, such as at least about 8000U/g, such as at least about
The xylanase activity of 8500U/g.
Enzymatic compositions according to the present invention can have cellulolytic activity.The systematic name of cellulase is 4- (1,3；
Isosorbide-5-Nitrae)-callose, 4- glucan hydrolase and cellulolytic enzyme or cellulase are classified in EC 3.2.1.4.Fine
Such as (1 → 4)-β-D- glucoside bond in cellulose, lichenin and corn callose is carried out interior cut by the plain enzyme of dimension
Hydrolysis, and also the Isosorbide-5-Nitrae-key in the callose also containing 1,3- key can be hydrolyzed.Cellulase also has other titles, such as
Inscribe-Isosorbide-5-Nitrae-callose enzyme, β-Isosorbide-5-Nitrae-glucanase, β-Isosorbide-5-Nitrae-endoglucan hydrolase, cellulase A,
Cellulosin AP, endoglucanase D, alkali cellulose enzyme, cellulase A 3, cellulose dextromase, 9.5 celluloses
Enzyme, avicelase, pancellase SS and Isosorbide-5-Nitrae-(1,3；Isosorbide-5-Nitrae)-callose 4- glucan hydrolase.
In one aspect of the invention, the cellulase activity of the enzymatic compositions according to the present invention passes through hereinafter title
Under " algoscopy ", described " cellulase activity method " measures.
In a further aspect, the present invention relates to as by the algoscopy mensure described in embodiment with inscribe -1,3
(4) enzyme of -1,4 beta-glucanase activity.
" 1,4 beta-glucanase " used herein or " beta- glucanase " refers to inscribe -1 of EC 3.2.1.6, and 3 (4)-β -
Glucanase, this enzyme is catalyzed (1- the ＞ 3)-key of callose or the interior cut water of (1- ＞ 4)-key in the case where there
Solution：When its reproducibility group participates in when the glucose residue of the key hydrolyzing originally is substituted at C-3.Will with according to this
The suitable 1,4 beta-glucanase that the enzyme of bright display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity is applied in combination include WO2004087889,
WO2005059084、WO9414953、WO2007056321、WO9531533、WO08023060、WO2005100582、
Described in WO9828410, WO9742301, WO2006066582, WO05118769, WO2005003319 and WO10059424
Any 1,4 beta-glucanase.
The algoscopy of standard is carried out under pH 5.0, or it can carry out under different pH value other to carry out to enzyme
Characterize and explanation.
Inscribe -1 of one unit, 3 (4) -1,4 beta-glucanase activities are defined as (pH 5.0 under conditions of this algoscopy
(or such as specified) and 50 DEG C) enzyme amount producing 1 micromoles glucose equivalent per minute.
In some embodiments, had as by the algoscopy institute described in embodiment according to the enzymatic compositions of the present invention
Measurement at least about 10000U/g, such as at least about 12000U/g, such as at least about 14000U/g, such as at least about 15000U/g, such as at least
The 1,4 beta-glucanase activity of about 18000U/g.
In a further aspect, according to the enzymatic compositions of the present invention have laminarin enzymatic activity or comprise any one or
The multiple other enzyme with laminarin enzymatic activity of person.Thallus Laminariae (Thallus Eckloniae) as described in " algoscopy " one section for the laminarin enzymatic activity is many
It is measured described by carbohydrase algoscopy.
Laminarinase can for classifying in inscribe -1 of E.C.3.2.1.6,3 (4) -1,4 beta-glucanases or classify in
Endoglucanase -1 of E.C.3.2.1.39,3- β-D- glucosidase.Inscribe -1,3 (4) -1,4 beta-glucanases, also known as laminarin
Enzyme, inscribe -1,3- 1,4 beta-glucanase, inscribe-Isosorbide-5-Nitrae -1,4 beta-glucanase, it is classified in E.C.3.2.1.6.It is many that substrate includes Thallus Laminariae (Thallus Eckloniae)
Sugar, lichenin and corn D- glucosan, this enzyme is catalyzed (1- ＞ 3)-key or (the 1- ＞ of callose in the case where there
4) the interior cut hydrolysis of-key：When the glucose residue that its reproducibility group participates in key to be hydrolyzed originally is substituted at C-3
When.Endoglucanase -1, cut hydrolytic enzyme, inscribe -1 in 3- β-D- glucosidase, also known as (1- ＞ 3)-beta glucan, 3- β-Portugal
Dextranase and laminarinase, are classified in E.C.3.2.1.39, its hydrolysis such as laminarin, Paramylon and pachyman
(1- ＞ 3)-β-D- glucoside bond in (1- ＞ 3)-callose in substrate.
In some respects, according to the enzymatic compositions of the present invention have Arabinanase activity or comprise another have Ah
Draw the enzyme of primary enzyme.Arabanase is classified as EC 3.2.1.99.Systematic name is 5- α-L- arabinan
5- α-L- arabinan hydrolytic enzyme, but it has other titles several, such as arabinan inscribe -1,5- α-L-arabinose
Glycosides enzyme and inscribe -1,5- α-L- arabanase, inscribe-α -1,5- arabanase, inscribe-arabanase, 1,
5- α-L- arabinan and 1,5- α-L- arabinan hydrolytic enzyme.Arabinase is by (1 → 5)-arabinan
(1 → 5)-α-arabinofuranosidase glycosidic bond carries out interior cut hydrolysis.Arabanase also acts on arabinan.
In one aspect of the invention, the arabinose enzymatic activity of the enzymatic compositions according to the present invention passes through hereafter title
Described arabinose enzyme assay under " algoscopy " measures.This algoscopy can be used using sugar at pH 3.5 and 50 DEG C
Radix Betae arabinan is carried out as substrate, and it can carry out under different pH and Temperature numerical other to carry out to enzyme
Characterize and explanation.At the 540nm of time per unit, absorbance incrementss calculate enzymatic activity.
The arabinase activity definition of one unit is that (pH 3.5 and 50 DEG C) produce Δ under conditions of this algoscopy
OD540nm.min-1Increase enzyme amount (being normalized with respect to total volume that measures).
In some respects, according to the enzymatic compositions of the present invention, there is β-D- glucoside glucohydralase activity or comprise another
One enzyme with β-D- glucoside glucohydralase activity.β-D- glucoside glucohydralase refers to the enzyme of E.C 3.2.1.21.
In some respects, according to the enzymatic compositions of the present invention, there is xylobiase activity or comprise another there is β-wood
The enzyme of glycosidase activity." xylobiase " or " xylan Isosorbide-5-Nitrae-xylobiase " refers to the enzyme of E.C 3.2.1.37.β-wood
The hydrolysis of glucosides enzyme catalysiss (1- ＞ 4)-β-D- xylan, to remove D- xylose residues successively from non-reducing end.
At some aspects of the present invention, according to the enzymatic compositions of the present invention, there is cellobiohydrolase activity or comprise
Another enzyme with cellobiohydrolase activity." cellobiohydrolase " or " cellulose Isosorbide-5-Nitrae-beta fibers bioside enzyme "
Refer to the enzyme of EC 3.2.1.91.Isosorbide-5-Nitrae-β-D- Portugal in cellulose Isosorbide-5-Nitrae-beta fibers bioside enzyme catalysiss cellulose and cellotetrose
The hydrolysis of glycosidic bond, discharges cellobiose from the non-reducing end of sugar chain.
The cellobiose enzymatic activity of the enzymatic compositions according to the present invention is by described fibre under hereafter title " algoscopy "
Dimension disaccharide enzyme assay measures.The algoscopy of standard is carried out under pH 5.0, and it can be carried out under different pH value
So that other sign and explanation are carried out to enzyme.
The cellobiohydrolase activity of one unit is defined as under conditions of this algoscopy that (pH 5.0 is (or as institute
Specify) and 50 DEG C) enzyme producing 1 micromolar paranitrophenol from p-nitrophenyl β-D- pyrans cellobioside per minute
Amount.
In some respects, according to the enzymatic compositions of the present invention, there is α-N- nofuranosidase activity or comprise another
One enzyme with nofuranosidase activity." α-N- arabinofuranosidase " or " alpha-N- arabinofuranosidase
Glycosidase " refers to the enzyme of EC 3.2.1.55.The end that α-N- arabinofuranosidase is catalyzed in α-L-arabinose glycosides is non-reduced
The hydrolysis of property α-L- arabinofuranosidase glucosides residue.
In one aspect of the invention, the nofuranosidase activity of the enzymatic compositions according to the present invention passes through hereafter
Under title " algoscopy ", described arabinofuranosidase algoscopy measures.This standard assay can be in pH 5.0 He
Carry out at 50 DEG C, and it can be carried out to carry out other sign and explanation to enzyme under different pH and Temperature numerical.
α-N- the nofuranosidase activity of one unit is defined as (pH 5.0 and 50 under conditions of this algoscopy
DEG C (or as specified)) per minute produce 1 micromolar paranitrophenol from p-nitrophenyl α-L- arabinofuranosidase glucosides
Enzyme amount.
In some respects, according to the enzymatic compositions of the present invention, there is glucosan Isosorbide-5-Nitrae-beta-glucosidase activity or comprise another
One enzyme with glucosan Isosorbide-5-Nitrae-beta-glucosidase activity." glucosan Isosorbide-5-Nitrae-β-glucosyl enzym " or " glucosan Isosorbide-5-Nitrae-β-Portugal
Glycosidase " refers to the enzyme of E.C3.2.1.74.Glucosan Isosorbide-5-Nitrae-β-glucosyl enzym is catalyzed (the 1- ＞ in (1- ＞ 4)-callose
4) hydrolysis of-key is to remove glucose unit successively.
In some respects, according to the enzymatic compositions of the present invention, there is the circumscribed-β of xyloglucan specificity-Isosorbide-5-Nitrae-glucosan
Enzymatic activity or the enzyme comprising the circumscribed-β of another xyloglucan specificity-Isosorbide-5-Nitrae-dextranase activity." xyloglucan specificity
Circumscribed-β-Isosorbide-5-Nitrae-glucanase " refers to the enzyme of E.C3.2.1.155.Circumscribed-the β of xyloglucan specificity-Isosorbide-5-Nitrae-glucanase is urged
Change the outer cut hydrolysis of (the 1- ＞ 4)-β-D- glucoside bond in xyloglucan.
Enzyme according to aforementioned aspects and enzymatic compositions can include reducing the viscosity of the aqueous solution comprising glucidtemnss
Technique in use.
Described enzyme and enzymatic compositions also can use in including the technique of aqueous solution of the starch-containing hydrolysate of bag filter.One
In a little embodiments, the aqueous solution comprising glucidtemnss is the converted mash for manufacturing medicated beer, in other embodiments, bag
The aqueous solution of starch-containing hydrolysate is food compositions.
Alternatively, can be used for producing fruit juice, wine, grain processing, combustion according to the enzymatic compositions of the present invention
Expect alcohol, the first generation or second filial generation bio-fuel such as bio-ethanol and drink alcohol.
In some embodiments, the first generation or second filial generation bio-fuel such as bio-ethanol from agricultural raw material such as Caulis Sacchari sinensis,
Rhizoma Solani tuber osi, Semen Maydiss, Semen Tritici aestivi, Sorghum vulgare Pers. etc. produce, or from fibrous material such as corn straw, switchgrass or other plant material
Produce.In both cases, become alcohol from raw material extraction fermentable sugars and by fermentable, alcohol is distilled off, and can be used as
Transport fuel.Can be used for this production of bio-fuel according to the enzymatic compositions of the present invention.Can add multienzyme complex with strengthen from
Raw material extracts polysaccharide, helps for polysaccharide to be degraded into fermentable sugars and/or strengthens machined parameters such as liquid from the separation of solid content, stream
Dynamic characteristic and aid pumpability.
The method of the present invention can be applicable to the converted mash preparation of any flour (grist).According to the present invention, flour can comprise
Any starch-containing and/or sugared vegetable material, described vegetable material may originate from any plant and plant part, including tuber, root,
Stem, leaf and seed.
In some embodiments, flour comprises grain, Tathagata from Fructus Hordei Vulgaris, Semen Tritici aestivi, rye (Secale cereale L.), Herba bromi japonici, Semen Maydiss, Oryza sativa L., buy
The grain of Luo Gaoliang, millet and Sorghum vulgare Pers., and it is highly preferred that at least 10% or more preferably at least 15%, even more desirably at least
25% or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or the wheat of even 100% (w/w)
The flour of bud juice is derived from grain.
In some embodiments, flour comprises the grain germinateing, such as barley malt.Preferably, at least 10% or more excellent
Choosing at least 15%, even more desirably at least 25% or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or
Even 100% (w/w)) the flour of beerwort be derived from the grain germinateing.
Term " converted mash " is understood to aqueous starch size, for example, comprise barley malt, the Fructus Hordei Vulgaris of crushing crushing
And/or other adjuvant or combinations thereof, subsequently by its with water mixing with to be separated become beerwort+wheat poor.
Term " converted mash separates " is understood to for example pass through filtering (lautering) or converted mash filters
(filtration) beerwort is separated with wheat grain.
Term " beer filtration " is understood to such separating technology, that is, be still in yeast cells in medicated beer and other
The material causing turbidity for example passes through microfiltration or membrane process removal.
Enzyme preparation, the such as enzyme preparation of food composition form prepared in accordance with the present invention, can be form or the work of solution
For solid, this depends on purposes and/or application model and/or mode of administration.Solid form can be used as being dried enzyme powder or work
For granulating enzyme.
In one aspect, the present invention provides and comprises enzyme according to the present invention or enzymatic compositions, zymophore and optional steady
Determine the enzymatic compositions preparation of agent and/or preservative.
In an additional aspect of the present invention, zymophore is selected from glycerol or water.
In yet another aspect, preparation comprises stabilizer.In one aspect, stabilizer is selected from inorganic salts, polyalcohols, sugar
Class and combinations thereof.In one aspect, stabilizer is inorganic salt such as potassium chloride.In yet another aspect, polyhydric alcohol be glycerol, third
Glycol or Sorbitol.In yet other aspects, sugar is small molecule carbohydrate, specifically several pleasantly sweet little
Molecule carbohydrate such as any one of glucose, Fructose and sucrose.
In yet another aspect, preparation comprises preservative.In one aspect, preservative is methyl parahydroxybenzoate, right
The preservative of nipasol, benzoate, sorbate or other food approved or their mixture.
Specific embodiments of the present invention.
In some embodiments, the enzyme of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity, optionally with any or
Multiple 1,4 beta-glucanase combinations according to the present invention, are provided that and brewage significantly reduced viscosity in application, thus contributing to improving
Converted mash separates with medicated beer.
The preferable xylanase characteristic brewageing application includes the one or more in following aspect：
A) zymolyte specificity
ο WE-AX/WU-AX ratios on viscosity has impact.In some embodiments, this ratio less than about 7.0, such as
Less than about 6.5, such as less than about 6.0, such as less than about 5.5, such as less than about 5.0, such as less than about 4.5.
B) zymolyte selectivity
The branch point that ο and enzyme are cut has how close it is believed that having impact to feature.
C) enzyme heat stability
The continuous-dissolution of AX (araboxylan)-heat stability key feature in ο converted mash preparation process.Therefore, exist
In some embodiments, the enzyme of the display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity according to the present invention is within the temperature range of 65-78 DEG C
It is heat-staple.
D) enzyme optimum pH.Therefore, in certain embodiments, the enzyme of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity has
Optimum pH in the range of pH 5.4-5.6.
E) enzyme level (being for example known key factor for xylanase)
Described brewage significantly reduced viscosity in application, can be measured as with the same terms and quantity use have
Know the comparison of enzyme or enzyme combined activity (such asMax) compare the viscosity reducing in brewageing application.
In some embodiments, the enzyme according to the display inscribe-Isosorbide-5-Nitrae-beta-xylanase of present invention activity, optionally with
Any or multiple 1,4 beta-glucanase combination according to the present invention, can improve converted mash in brewageing application and medicated beer divides
From.
In certain embodiments, the enzyme of the display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity according to the present invention, optionally with root
According to any or multiple 1,4 beta-glucanase combination of the present invention, abnormal flavour can be made to be formed such as related to araboxylan decomposition
Abnormal flavour formed Potential feasibility low.
In certain embodiments, the enzyme of the display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity according to the present invention, optional and any
One or more, according to the 1,4 beta-glucanase combination of the present invention, can make the risk reduction of filter bed collapse (as in filtering).
In certain embodiments, the enzyme of the display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity according to the present invention, optional and any
One or more can make the Potential feasibility minimizing of abnormal flavour and/or the shape of abnormal flavour according to the 1,4 beta-glucanase combination of the present invention
Become to reduce.One aspect of the present invention is related to show the enzyme of inscribe-Isosorbide-5-Nitrae-beta-xylanase activity, this enzyme comprises and selected from SEQ
ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID
NO：17 and SEQ ID NO：18 any one or their any function fragment has the aminoacid sequence of at least 80% homogeneity
Row.
It is related to show inscribe -1, the enzyme of 3 (4) -1,4 beta-glucanase activities on the other hand, this enzyme comprises and selected from SEQ ID
NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10 and SEQ ID NO：11、SEQ ID NO：12、SEQ ID
NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16 any one or their any function fragment have
The aminoacid sequence of at least 80% homogeneity.
In some embodiments of the present invention, the enzyme of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity to solubility I
The activity of primary xylan substrate (WE-AX) with to insoluble araboxylan substrate (WU-AX) araboxylan substrate
The ratio of activity less than about 7.0, such as less than about 6.5, such as less than about 6.0, such as less than about 5.5, such as less than about 5.0, such as less than about
4.5.
In some embodiments, the optimum temperature of the enzyme according to the present invention is in the range of 40-70 DEG C, such as in 45-65
In the range of DEG C, such as in the range of 50-65 DEG C, such as in 55-65 DEG C of scope.
In some embodiments, the enzyme according to the present invention with selected from SEQ ID NO：Arbitrary aminoacid sequence of 1-18 or
Their any function fragment of person has at least 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,
97th, 98 or 99% homogeneity.
In some embodiments, the aminoacid sum of the enzyme according to the present invention less than 350, such as less than 340, such as less than
330th, such as less than 320, such as less than 310, such as less than 300 aminoacid, such as in the range of 200-350 aminoacid, such as exists
In the range of 220-345 aminoacid.
In some embodiments, the aminoacid sequence of enzyme according to the present invention with selected from SEQ ID NO：1-18's
Arbitrary aminoacid sequence or their any function fragment compare have at least one, two, three, four, five, six
Individual, seven, eight, nine or ten aminoacid replacement.
In some embodiments, the aminoacid sequence of enzyme according to the present invention with selected from SEQ ID NO：1-18's
Arbitrary aminoacid sequence or their any function fragment compare have most one, two, three, four, five, six
Individual, seven, eight, nine or ten aminoacid replacement.
In some embodiments, SEQ ID NO is comprised according to the enzyme of the present invention：The appointing of any one or they of 1-18
What aminoacid sequence determined by function fragment.
In some embodiments, the enzyme according to the present invention is by SEQ ID NO：Any one or they of 1-18 any
Aminoacid sequence composition determined by function fragment.
Another importance of the present invention is related to comprise the display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity according to the present invention
Enzyme and any or multiple 1,4 beta-glucanases the compositionss combining.In some embodiments, this is a kind of or many
Planting 1,4 beta-glucanase is according to the present invention.
Another importance of the present invention is display inscribe -1 comprising according to the present invention, 3 (4)-beta glucan enzyme activity
The enzyme of property and the compositionss combining of any or multiple xylanase.In some embodiments, this is a kind of or many
Plant the enzyme of the display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity that xylanase is according to the present invention.In some embodiments, this
Plant or multiple xylanase is according to SEQ ID NO：17 and/or SEQ ID NO：18 or their any function fragment
Enzyme.
In some embodiments, the enzyme of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity and display inscribe -1,3 (4)-β -
The combination of the enzyme of dextranase activity is according to following table：
It should be understood that the first enzyme is that display inscribe-Isosorbide-5-Nitrae-the active enzyme of beta-xylanase can in any one combinations thereof
With a kind of display inscribe -1, the enzyme of 3 (4) -1,4 beta-glucanase activities is combined, the ratio between two kinds of enzymes is 1: 10,2: 10,
3∶10、4∶10、5∶10、6∶10、7∶10、8∶10、9∶10、10∶10、10∶9、10∶8、10∶7、10∶6、10∶5、10∶4、10∶3、
10: 2 or 10: 1, such as 1: in the range of 10-10: 1, such as 2: 10-10: 2, such as 3: 10-10: 3, such as 4: 10-10: 4, such as 5:
10-10: 5, such as 6: 10-10: 6, such as 7: 10-10: 7, such as 8: 10-10: 8, or 9: in the range of 10-10: 9.
In some embodiments, comprise the group selected from least two enzymes set forth below according to the compositionss of the present invention
Close, or comprise its aminoacid sequence and there is at least 80% sequence together with corresponding SEQ ID or their any function fragment
The combination of at least two enzymes of one property：
SEQ ID NO：1 and SEQ ID NO：7；
SEQ ID NO：2 and SEQ ID NO：7；
SEQ ID NO：3 and SEQ ID NO：7；
SEQ ID NO：4 and SEQ ID NO：7；
SEQ ID NO：5 and SEQ ID NO：7；
SEQ ID NO：6 and SEQ ID NO：7；
SEQ ID NO：17 and SEQ ID NO：7；
SEQ ID NO：18 and SEQ ID NO：7；
SEQ ID NO：1 and SEQ ID NO：8；
SEQ ID NO：2 and SEQ ID NO：8；
SEQ ID NO：3 and SEQ ID NO：8；
SEQ ID NO：4 and SEQ ID NO：8；
SEQ ID NO：5 and SEQ ID NO：8；
SEQ ID NO：6 and SEQ ID NO：8；
SEQ ID NO：17 and SEQ ID NO：8；
SEQ ID NO：18 and SEQ ID NO：8；
SEQ ID NO：1 and SEQ ID NO：9；
SEQ ID NO：2 and SEQ ID NO：9；
SEQ ID NO：3 and SEQ ID NO：9；
SEQ ID NO：4 and SEQ ID NO：9；
SEQ ID NO：5 and SEQ ID NO：9；
SEQ ID NO：6 and SEQ ID NO：9；
SEQ ID NO：17 and SEQ ID NO：9；
SEQ ID NO：18 and SEQ ID NO：9；
SEQ ID NO：1 and SEQ ID NO：10；
SEQ ID NO：2 and SEQ ID NO：10；
SEQ ID NO：3 and SEQ ID NO：10；
SEQ ID NO：4 and SEQ ID NO：10；
SEQ ID NO：5 and SEQ ID NO：10；
SEQ ID NO：6 and SEQ ID NO：10；
SEQ ID NO：17 and SEQ ID NO：10；
SEQ ID NO：18 and SEQ ID NO：10；
SEQ ID NO：1 and SEQ ID NO：11；
SEQ ID NO：2 and SEQ ID NO：11；
SEQ ID NO：3 and SEQ ID NO：11；
SEQ ID NO：4 and SEQ ID NO：11；
SEQ ID NO：5 and SEQ ID NO：11；
SEQ ID NO：6 and SEQ ID NO：11；
SEQ ID NO：17 and SEQ ID NO：11；
SEQ ID NO：18 and SEQ ID NO：11；
SEQ ID NO：1 and SEQ ID NO：12；
SEQ ID NO：2 and SEQ ID NO：12；
SEQ ID NO：3 and SEQ ID NO：12；
SEQ ID NO：4 and SEQ ID NO：12；
SEQ ID NO：5 and SEQ ID NO：12；
SEQ ID NO：6 and SEQ ID NO：12；
SEQ ID NO：17 and SEQ ID NO：12；
SEQ ID NO：18 and SEQ ID NO：12；
SEQ ID NO：1 and SEQ ID NO：13；
SEQ ID NO：2 and SEQ ID NO：13；
SEQ ID NO：3 and SEQ ID NO：13；
SEQ ID NO：4 and SEQ ID NO：13；
SEQ ID NO：5 and SEQ ID NO：13；
SEQ ID NO：6 and SEQ ID NO：13；
SEQ ID NO：17 and SEQ ID NO：13；
SEQ ID NO：18 and SEQ ID NO：13；
SEQ ID NO：1 and SEQ ID NO：14；
SEQ ID NO：2 and SEQ ID NO：14；
SEQ ID NO：3 and SEQ ID NO：14；
SEQ ID NO：4 and SEQ ID NO：14；
SEQ ID NO：5 and SEQ ID NO：14；
SEQ ID NO：6 and SEQ ID NO：14；
SEQ ID NO：17 and SEQ ID NO：14；
SEQ ID NO：18 and SEQ ID NO：14；
SEQ ID NO：1 and SEQ ID NO：15；
SEQ ID NO：2 and SEQ ID NO：15；
SEQ ID NO：3 and SEQ ID NO：15；
SEQ ID NO：4 and SEQ ID NO：15；
SEQ ID NO：5 and SEQ ID NO：15；
SEQ ID NO：6 and SEQ ID NO：15；
SEQ ID NO：17 and SEQ ID NO：15；
SEQ ID NO：18 and SEQ ID NO：15；
SEQ ID NO：1 and SEQ ID NO：16；
SEQ ID NO：2 and SEQ ID NO：16；
SEQ ID NO：3 and SEQ ID NO：16；
SEQ ID NO：4 and SEQ ID NO：16；
SEQ ID NO：5 and SEQ ID NO：16；
SEQ ID NO：6 and SEQ ID NO：16；
SEQ ID NO：17 and SEQ ID NO：16；With
SEQ ID NO：18 and SEQ ID NO：16.
In some embodiments, inscribe -1,3 (4) -1,4 beta-glucanase activities and inscribe-Isosorbide-5-Nitrae-beta-xylanase activity
Spread out from least two different enzymes, such as from least two different enzymes of two kinds of different plant species.
In some embodiments, using during according to the compositionss of the present invention before carrying out filtering in brewageing application,
Gross pressure raises and is reduced to less than 470mm WC, such as less than 450mm WC, such as less than 430mm WC, such as less than 410mm WC, such as
Less than 390mm WC, such as less than 370mm WC, such as less than 350mm WC, such as less than 330mm WC, such as less than 310mm WC, such as little
Numerical value in 300mm WC, such as less than 290mm WC.
In some embodiments, before carrying out filtering in brewageing application using described compositionss when, and using not having
The negative control having described compositionss is compared, gross pressure raise reduce at least 5,7,9,11,13,15,17,19,21,23,25,
27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、
77th, 79,81,83,85,87,89,91,93 or 95%.
In some embodiments, using the combination according to the present invention before carrying out beerwort separation in brewageing application
During thing, with respect to the comparison not having enzyme, beerwort filterability (the wort volume measurement by collecting after filtering 5 minutes) carries
Up to more than 1.5, such as more than 1.6, such as more than 1.7, such as more than 1.8, such as more than 1.9, such as more than 2.0, such as more than 2.1, such as super
Cross 2.2, such as more than 2.3, such as more than 2.4, such as more than 2.5.
In some embodiments, with using compared with not having the negative control of described compositionss, beerwort filterability is (logical
Filter 5 minutes after collect wort volume measurement) improve at least 5,7,9,11,13,15,17,19,21,23,25,27,
29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、
79、81、83、85、87、89、91、93、95、97、99、110、120、130、140、150、160、170、180、190、200、210、
220th, 230,240,250,260,270,280,290 or 300%.
In some embodiments, comprise any or multiple other enzyme according to the compositionss of the present invention.One
In a little embodiments, this one or more other enzyme is selected from set forth below：Classify in EC 3.2.1.32, EC
3.2.1.136 or the xylanase of EC 3.2.1.156, cellulase, laminarinase, inscribe -1,5- α-L- is Arabic poly-
Carbohydrase, β-D- glucoside glucohydralase, xylobiase, cellobiohydrolase, glucosan Isosorbide-5-Nitrae-β-glucosyl enzym, wooden Portugal
Circumscribed-the β of polysaccharide specificity-Isosorbide-5-Nitrae-glucanase and α-N- arabinofuranosidase.
Being used alone shown in sequence as herein described and according to the present invention or being combined with other enzymes or compound is made
Sequence and enzyme, can be with and without signal peptide.
Algoscopy
DNS cellulase activity method (DNS CMC method)
Systematic name：Isosorbide-5-Nitrae-(1,3；Isosorbide-5-Nitrae)-callose 4- glucan hydrolase
IUB numbers：EC 3.2.1.4
Principle
The algoscopy of cellulase be based on to the Isosorbide-5-Nitrae-β in carboxymethyl cellulose (CMC) (a kind of β-Isosorbide-5-Nitrae-glucosan)-
Cut hydrolysis in the enzymatic of D- glycosidic bond.The product (β-Isosorbide-5-Nitrae glucosan oligosaccharide) of this reaction is by using 3,5- dinitrosalicylic
Acid reagent measures the corresponding rising of reproducibility group and carries out colorimetric determination.(worked as with glucose by the concentration of reproducibility group
Gauge) and 540nm at absorbance between relation calculate enzymatic activity.
This algoscopy is carried out under pH 5.0, but it can carry out under different pH value with enzyme is carried out other sign and
Explanation.
Unit definition
The cellulase activity of one unit be defined as under conditions of this algoscopy (pH 5.0 (or as specified) and
50 DEG C) under per minute produce 1 micromolar glucose equivalent enzyme amount.
Material
Carboxymethyl cellulose.Supplier：Megazyme company limited.Production code member：CM-Cellulose 4M
D-Glucose ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10117.Molecular weight：
180.16
Anhydrous sodium acetate ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10236.Molecular weight：
82.03
Acetic acid (" glacial acetic acid ") ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10001.Molecule
Amount：60.05
3,5- dinitrosalicylic acid GPR (3,5- dinitros -2 hydroxybenzoic acid).Supplier：Merck company limited
(BDH).Production code member：28235
Sodium hydrate particle ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10252.Molecular weight：
40.00
(+)-sodium potassium tartrate tetrahydrate ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10219.Molecule
Amount：282.22
Solution in 0.1M sodium acetate buffer for 1.5% (weight/volume solution) carboxymethyl cellulose (CMC), pH
5.0 (substrate solutions).
3,5- dinitrosalicylic acids (DNS) solution.The DNS of 20g/L is in containing 32g/L sodium hydrate particle and 600g/L
In the buffer of (+)-sodium potassium tartrate tetrahydrate.
Glucose standards solution (0.50mg/ml)
Program
Enzymatic compositions are diluted in sample, using the concentration of glucose system of 0,0.125,0.25,0.375 and 0.5mg/ml
Make the glucose standard curve shown in Fig. 2.
The enzymatic solution of 0.25ml is mixed at 50 DEG C with the substrate solution (1.5% weight/volume) of 1.75ml, 10 minutes
Pass through afterwards to add DNS solution stopped reaction.It is then heated to 95 DEG C to keep 5 minutes.
Different optical density (the OD at 540nm for the sample of measurement540nm).
Calculate
Standard curve determination enzymatic activity shown in from Fig. 2.
Activity is calculated as below：
Wherein：
T=Δ OD540nmTest
=OD540nmTest-OD540nmBlank
The slope (about 1.0) of m=standard curve
The y intercept (always negative, about -0.02) of c=standard curve
180.16 the molecular weight of ≡ glucose
103≡ is scaled micromole
A ≡ measures volume (ml)
V ≡ enzyme volume (ml)
T ≡ minute (minute)
D=actual enzyme extension rate (for example 1 liter being diluted to for 1.000g, D=1000)
Laminarinase (DNS laminarin method)
Principle
It is related to the interior cut hydrolysis of 1, the 3- glycosidic bond in 1,3- callose by this reaction of laminarin enzyme catalysiss.
Substrate includes laminarin, Paramylon and pachyman.The product (β -1,3- glucosan oligosaccharide) of this reaction is by using 3,5-
Edlefsen's reagent measures the corresponding rising of reproducibility group and carries out colorimetric determination.Concentration by reproducibility group
Relation between absorbance at (in terms of glucose equivalent) and 540nm calculates enzymatic activity.
This algoscopy is carried out at pH 5.0 and 50 DEG C, but it can be carried out to enter to enzyme under different pH and Temperature numerical
The other sign of row and explanation.
Unit definition
The laminarinase activity definition of one unit is that (pH 5.0 and 50 DEG C are (or such as under conditions of this algoscopy
Specified)) under per minute produce 1 micromolar glucose equivalent enzyme amount.
Material
See above the material that cellulase activity algoscopy is provided.
Laminarin (from Laminaria digitata (Laminaria digitata)).Supplier：The limited duty of Sigma-Aldrich
Ren company.Production number：L 9634
1.00% (weight/volume solution) laminarin solution (substrate solution 0.1M sodium acetate buffer, pH 5.0)
The enzymatic solution that 1.75ml laminarin solution is diluted with 0.25ml mixes 10 minutes at 50 DEG C, by adding
2ml DNS solution stopped reaction.
Make standard curve using 0,0.125,0.25,0.5 and 0.75mg/ml glucose solution.
Optical density (OD at measurement 540nm540nm).
Calculate
Activity is calculated as below：
Wherein：
T=Δ OD540nmTest
=OD540nmTest-OD540nmBlank
The slope (about 1.0) of M=standard curve
The y intercept (always negative, about -0.03) of C=standard curve
180.16 the molecular weight of ≡ glucose
103≡ is scaled micromole
A ≡ measures volume (ml)
V ≡ enzyme volume (ml)
T ≡ minute (minute)
D=enzyme extension rate (is for example diluted to 1 liter for 1g, D=1000)
Arabinose enzymatic determination.
Principle
The algoscopy of arabinose enzymatic activity is based upon using 3,5- edlefsen's reagent measurement reproducibility group
Corresponding increase and carry out colorimetric determination.By the concentration (gauge is worked as with arabinose) of reproducibility group and the extinction of 540nm
Relation between degree calculates enzymatic activity.
This algoscopy is carried out under pH 3.5, but it can carry out under different pH value with enzyme is carried out other sign and
Explanation.
Unit definition
Arabinase (arabanase (inscribe -1,5- α-L- the arabanase)) activity definition of one unit
It is generation per minute 1 micromolar arabinose under under conditions of this algoscopy (pH 3.5 (or such as specified) and 50 DEG C)
The enzyme amount of equivalent.
Material
Megazyme sugar beet araban
Arabinose Sigma A3131 molecular weight：150.1
Anhydrous sodium acetate ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10236.Molecular weight：
82.03
Acetic acid (" glacial acetic acid ") ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10001.Molecule
Amount：60.05
3,5- dinitrosalicylic acid GPR (3,5- dinitros -2 hydroxybenzoic acid).Supplier：Merck company limited
(BDH).Production code member：28235
Sodium hydrate particle ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10252.Molecular weight：
40.00
(+)-sodium potassium tartrate tetrahydrate ' AnalaR '.Supplier：Merck company limited (BDH).Production code member：10219.Molecule
Amount：282.22
1.5% (weight/volume solution) arabinan solution in 0.1M sodium acetate buffer, pH 3.5 (substrate
Solution).
3,5- dinitrosalicylic acids (DNS) solution.The DNS of 20g/L is in containing 32g/L sodium hydrate particle and 600g/L
In the buffer of (+)-sodium potassium tartrate tetrahydrate.
Arabinose standard solution (0.50mg/ml)
Program
Enzymatic compositions are diluted in sample, using the arabinose concentrations of 0,0.125,0.25,0.375 and 0.5mg/ml
Make arabinose standard curve.
The enzymatic solution of 0.25ml is mixed at 50 DEG C with the substrate solution (1.5% weight/volume) of 1.75ml, 10 minutes
Pass through afterwards to add DNS solution stopped reaction.It is then heated to 95 DEG C to keep 5 minutes.
Different optical density (the OD at 540nm for the sample of measurement540nm).
Calculate
From standard curve determination enzymatic activity.
Activity is calculated as below：
Wherein：
T=Δ OD540nmTest
=OD540nmTest-OD540nmBlank
The slope (about 1.0) of m=standard curve
The y intercept (always negative, about -0.02) of c=standard curve
150.13 the molecular weight of ≡ arabinose
103≡ is scaled micromole
A ≡ measures volume (ml)
V ≡ enzyme volume (ml)
T ≡ minute (minute)
D=actual enzyme extension rate (for example 1 liter being diluted to for 1.000g, D=1000)
Arabinofuranosidase algoscopy.
By α-N- arabinofuranosidase catalysis reaction be related to α-L-arabinose glycosides irreducibility α-L- I
The hydrolysis of the end key at primary furanoside residue.This enzyme effect in α-L- arabinofuranosidase glucosides, containing (1,3)-key and/or
α-L- the arabinan of (1,5)-key, araboxylan and arabinogalactan.
The algoscopy of α-N- arabinofuranosidase is the enzymatic based on p-nitrophenyl α-L- arabinofuranosidase glucosides
Hydrolysis.This algoscopy is " 2 points " method, rather than " continuous monitoring " method.The calculating of enzymatic activity be based on only during incubating start and
At the end of obtain measured value.Product paranitrophenol is by colorimetric method for determining (after adjustment pH).By paranitrophenol
Concentration and 400nm at absorbance between relation calculate enzymatic activity.
The preparation of the enzymatic solution of dilution：
Prepare whole enzymatic solution with glass distilled water from powder or liquid enzyme formulation.By avoid being related to small size or
The big dilution step of person's weight makes algoscopy dilution error reduce to minimum.When preparing enzyme diluent, it is more exact that weighing up just
Beginning enzyme sample, is also such even with fluid sample.If it does, in the case of fluid sample it is therefore necessary to
Measure proportion at 20 DEG C for this liquid.
Because this algoscopy is " 2 points " method rather than " continuous monitoring " method it is important that guaranteeing different enzyme systems and condition
Incubation during interior linear.It has therefore proved that this mensure under the standard assay conditions of concentration of substrate, pH, temperature and minute
Method is in Δ OD540nmIt is linear in the range of test (T)=0.20-1.50.But, in order to implement well, this algoscopy exists
ΔOD540nmThe restriction range of operation of test (T)=0.400-0.800.
Program
Each enzyme sample measures and is related to three analyses：Retest (" test ") analysis and blank (" blank ") analysis.Institute
The analysis of the single enzyme sample of program description being given.
Add the enzymatic solution of 0.25ml dilution to each solution at 50 DEG C, pass through after 10 minutes to add the sweet ammonia of 0.4M of 4ml
Acid solution pH 10.8 (termination reagent) terminating reaction.
With water for blank measure absorbance at 400nm at 25 DEG C.
Measure the OD of measured retest400nmTest；
Measure OD400nmBlank.
Calculate
ΔOD400nmTest (T)=OD400nmTest -0D400nmBlank
Wherein：T=OD400nm test-OD400nm is blank
The molar extinction coefficient (1cm optical path length) of 18300=paranitrophenol
V=7.25 (the total liquid volume (ml) in test)
T=10 (minute)
1u=1 μm of ol.min-1
E=0.25 (volume (ml) of the enzyme sample of dilution)
D=enzyme extension rate (is for example diluted to 1 liter for 1ml, D=1000)
Cellobiohydrolase algoscopy.
Principle
It is related to Isosorbide-5-Nitrae-β-D- glucoside bond in cellulose and cellotetrose by the reaction that cellobiohydrolase is catalyzed
Hydrolysis, discharges cellobiose from the non reducing end of sugar chain.
The algoscopy of cellobiohydrolase is the enzymatic hydrolysiss based on p-nitrophenyl β-D- pyrans cellobioside.Instead
Answer product paranitrophenol by colorimetric method for determining (after adjustment pH).By the concentration of paranitrophenol and the extinction of 400nm
Relation between degree calculates enzymatic activity.
This algoscopy is in Δ OD540nmThe linear restriction range of operation of test (T)=0.400-0.800.
Program
Each enzyme sample measures and is related to three analyses：Retest (test) analysis and blank (blank) analysis.Given
The single enzyme sample of program description analysis.
Add the enzymatic solution of 0.25ml dilution to the test solution at 50 DEG C, each pipe backward adds 4ml's within 30 minutes
0.4M glycine solution pH 10.8 (termination reagent).
In 1cm glass color comparison tube, with water as blank, measure absorbance at 20 DEG C at 400nm.
Measure the OD400nm test of measured retest；
Measure OD400nm blank.
Calculate
ΔOD400nmTest (T)=OD400nmTest-OD400nmBlank
Wherein：T=OD400nmTest-OD400nmBlank
The molar extinction coefficient (1cm optical path length) of 18300=paranitrophenol
V=7.25 (the total liquid volume (ml) in test)
1000=is scaled liter
106=it is scaled micromole
T=30 (minute)
1u=1 μm of ol.min-1
E=0.25 (volume (ml) of the enzyme sample of dilution)
D=enzyme extension rate (is for example diluted to 1 liter for 1ml, D=1000)
Numbering according to embodiment of the present invention：
1. the enzyme of a kind of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity, this enzyme comprises and selected from SEQ ID NO：1、SEQ
ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：17 and SEQ ID
NO：18 any one or their any function fragment has the aminoacid sequence of at least 80% homogeneity.
2. the enzyme according to embodiment 1, this enzyme to the activity of solubility araboxylan substrate (WE-AX) with right
The ratio of the activity of insoluble araboxylan substrate (WU-AX) araboxylan substrate less than about 7.0, such as less than about 6.5,
Such as less than about 6.0, such as less than about 5.5, such as less than about 5.0, such as less than about 4.5.
3. a kind of display inscribe -1, the enzyme of 3 (4) -1,4 beta-glucanase activities, this enzyme comprises and selected from SEQ ID NO：7、
SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10 and SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、
SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16 any one or their any function fragment have at least
The aminoacid sequence of 80% homogeneity.
4. the enzyme according to any one of embodiment 1-3, the optimum temperature of this enzyme in the range of 40-70 DEG C, such as
In the range of 45-65 DEG C, such as in the range of 50-65 DEG C, such as in the range of 55-65 DEG C.
5. the enzyme according to any one of embodiment 1-4, wherein said enzyme with selected from SEQ ID NO：1-18 appoints
Amino acid sequence or their any function fragment have at least 81,82,83,84,85,86,87,88,89,90,91,
92nd, 93,94,95,96,97,98 or 99% homogeneity.
6. the enzyme according to any one of embodiment 1-5, its total amino acidss number be less than 350, such as less than 340, such as
Less than 330, such as less than 320, such as less than 310, such as less than 300 aminoacid, such as in the range of 200-350 aminoacid, such as
In the range of 220-345 aminoacid.
7. the enzyme according to any one of embodiment 1-6, the aminoacid sequence of described enzyme with selected from SEQ ID NO：
Arbitrary aminoacid sequence of 1-18 or their any function fragment compare have at least one, two, three, four, five
Individual, six, seven, eight, nine or ten aminoacid replacement.
8. the enzyme according to any one of embodiment 1-7, the aminoacid sequence of described enzyme with selected from SEQ ID NO：
Arbitrary aminoacid sequence of 1-18 or their any function fragment compare have most one, two, three, four, five
Individual, six, seven, eight, nine or ten aminoacid replacement.
9. the enzyme according to any one of embodiment 1-8, this enzyme comprises SEQ ID NO：1-18 any one or
Their aminoacid sequence determined by any function fragment.
10. the enzyme according to any one of embodiment 1 or 3, this enzyme is by SEQ ID NO：1-18 any one or
Their composition of aminoacid sequence determined by any function fragment of person.
A kind of 11. DNA construct, it comprises to encode the DNA sequence of the enzyme according to any one of embodiment 1-10.
A kind of 12. expression vectors, it comprises the DNA construct according to embodiment 11.
A kind of 13. cells, it has used the carrier conversion of the DNA construct of embodiment 11 or embodiment 12.
A kind of 14. preparations, it comprises enzyme according to any one of embodiment 1-10 or according to embodiment 11
Described DNA construct or the carrier according to embodiment 12 or the cell according to embodiment 13.
A kind of 15. compositionss, its comprise display inscribe-Isosorbide-5-Nitrae-β according to any one of embodiment 1,2,4-10-
The enzyme of xylanase activity is combined with any or multiple 1,4 beta-glucanase.
16. compositionss according to embodiment 15, one or more 1,4 beta-glucanases wherein said are according to reality
Apply display inscribe -1 any one of scheme 3-10, the enzyme of 3 (4) -1,4 beta-glucanase activities.
A kind of 17. compositionss, it comprises display inscribe -1 according to any one of embodiment 3-10,3 (4)-β -
The enzyme of dextranase activity is combined with any or multiple xylanase.
18. compositionss according to claim 17, one or more xylanase wherein said are according to enforcement
Scheme 1,2, the enzyme of display inscribe-Isosorbide-5-Nitrae-beta-xylanase activity any one of 4-10.
19. compositionss according to any one of embodiment 15-18, wherein said inscribe -1,3 (4)-beta glucans
Enzymatic activity and described inscribe-Isosorbide-5-Nitrae-beta-xylanase activity are spread out from least two different enzymes, as from two kinds of different plant species
At least two different enzymes.
20. compositionss according to any one of embodiment 15-19, described compositionss comprise selected from set forth below
The combination of at least two enzymes, or comprise its aminoacid sequence and have with corresponding SEQ ID or their any function fragment
The combination of at least two enzymes of at least 80% sequence iden：
SEQ ID NO：1 and SEQ ID NO：7；
SEQ ID NO：2 and SEQ ID NO：7；
SEQ ID NO：3 and SEQ ID NO：7；
SEQ ID NO：4 and SEQ ID NO：7；
SEQ ID NO：5 and SEQ ID NO：7；
SEQ ID NO：6 and SEQ ID NO：7；
SEQ ID NO：17 and SEQ ID NO：7；
SEQ ID NO：18 and SEQ ID NO：7；
SEQ ID NO：1 and SEQ ID NO：8；
SEQ ID NO：2 and SEQ ID NO：8；
SEQ ID NO：3 and SEQ ID NO：8；
SEQ ID NO：4 and SEQ ID NO：8；
SEQ ID NO：5 and SEQ ID NO：8；
SEQ ID NO：6 and SEQ ID NO：8；
SEQ ID NO：17 and SEQ ID NO：8；
SEQ ID NO：18 and SEQ ID NO：8；
SEQ ID NO：1 and SEQ ID NO：9；
SEQ ID NO：2 and SEQ ID NO：9；
SEQ ID NO：3 and SEQ ID NO：9；
SEQ ID NO：4 and SEQ ID NO：9；
SEQ ID NO：5 and SEQ ID NO：9；
SEQ ID NO：6 and SEQ ID NO：9；
SEQ ID NO：17 and SEQ ID NO：9；
SEQ ID NO：18 and SEQ ID NO：9；
SEQ ID NO：1 and SEQ ID NO：10；
SEQ ID NO：2 and SEQ ID NO：10；
SEQ ID NO：3 and SEQ ID NO：10；
SEQ ID NO：4 and SEQ ID NO：10；
SEQ ID NO：5 and SEQ ID NO：10；
SEQ ID NO：6 and SEQ ID NO：10；
SEQ ID NO：17 and SEQ ID NO：10；
SEQ ID NO：18 and SEQ ID NO：10；
SEQ ID NO：1 and SEQ ID NO：11；
SEQ ID NO：2 and SEQ ID NO：11；
SEQ ID NO：3 and SEQ ID NO：11；
SEQ ID NO：4 and SEQ ID NO：11；
SEQ ID NO：5 and SEQ ID NO：11；
SEQ ID NO：6 and SEQ ID NO：11；
SEQ ID NO：17 and SEQ ID NO：11；
SEQ ID NO：18 and SEQ ID NO：11；
SEQ ID NO：1 and SEQ ID NO：12；
SEQ ID NO：2 and SEQ ID NO：12；
SEQ ID NO：3 and SEQ ID NO：12；
SEQ ID NO：4 and SEQ ID NO：12；
SEQ ID NO：5 and SEQ ID NO：12；
SEQ ID NO：6 and SEQ ID NO：12；
SEQ ID NO：17 and SEQ ID NO：12；
SEQ ID NO：18 and SEQ ID NO：12；
SEQ ID NO：1 and SEQ ID NO：13；
SEQ ID NO：2 and SEQ ID NO：13；
SEQ ID NO：3 and SEQ ID NO：13；
SEQ ID NO：4 and SEQ ID NO：13；
SEQ ID NO：5 and SEQ ID NO：13；
SEQ ID NO：6 and SEQ ID NO：13；
SEQ ID NO：17 and SEQ ID NO：13；
SEQ ID NO：18 and SEQ ID NO：13；
SEQ ID NO：1 and SEQ ID NO：14；
SEQ ID NO：2 and SEQ ID NO：14；
SEQ ID NO：3 and SEQ ID NO：14；
SEQ ID NO：4 and SEQ ID NO：14；
SEQ ID NO：5 and SEQ ID NO：14；
SEQ ID NO：6 and SEQ ID NO：14；
SEQ ID NO：17 and SEQ ID NO：14；
SEQ ID NO：18 and SEQ ID NO：14；
SEQ ID NO：1 and SEQ ID NO：15；
SEQ ID NO：2 and SEQ ID NO：15；
SEQ ID NO：3 and SEQ ID NO：15；
SEQ ID NO：4 and SEQ ID NO：15；
SEQ ID NO：5 and SEQ ID NO：15；
SEQ ID NO：6 and SEQ ID NO：15；
SEQ ID NO：17 and SEQ ID NO：15；
SEQ ID NO：18 and SEQ ID NO：15；
SEQ ID NO：1 and SEQ ID NO：16；
SEQ ID NO：2 and SEQ ID NO：16；
SEQ ID NO：3 and SEQ ID NO：16；
SEQ ID NO：4 and SEQ ID NO：16；
SEQ ID NO：5 and SEQ ID NO：16；
SEQ ID NO：6 and SEQ ID NO：16；
SEQ ID NO：17 and SEQ ID NO：16；With
SEQ ID NO：18 and SEQ ID NO：16.
21. compositionss according to any one of embodiment 15-20, wherein when carry out in brewageing application filtering it
Front using described compositionss when, gross pressure raise be reduced to less than 470mm WC, such as less than 450mm WC, such as less than 430mm
WC, such as less than 410mm WC, such as less than 390mm WC, such as less than 370mm WC, such as less than 350mm WC, such as less than 330mm
The numerical value of WC, such as less than 310mm WC, such as less than 300mm WC, such as less than 290mm WC.
22. compositionss according to any one of embodiment 15-21, wherein when carry out in brewageing application filtering it
Front using described compositionss when, and using compared with there is no the negative control of described compositionss, gross pressure raise reduce at least 5,7,
9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、
61st, 63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93 or 95%.
23. compositionss according to any one of embodiment 15-22, wherein when carrying out beerwort in brewageing application
When before separating using described compositionss, with respect to the comparison not having enzyme, by the wort volume institute collected after filtering 5 minutes
The beerwort filterability of measurement improve to more than 1.5, such as more than 1.6, such as more than 1.7, such as more than 1.8, such as more than 1.9, such as
More than 2.0, such as more than 2.1, such as more than 2.2, such as more than 2.3, such as more than 2.4, such as more than 2.5.
24. compositionss according to any one of embodiment 15-23, wherein when carrying out beerwort in brewageing application
When before separating using described compositionss, with using compared with there is no the negative control of described compositionss, receive by after filtering 5 minutes
The beerwort filterability measured by wort volume of collection improves at least 5,7,9,11,13,15,17,19,21,23,25,27,
29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、
79、81、83、85、87、89、91、93、95、97、99、110、120、130、140、150、160、170、180、190、200、210、
220th, 230,240,250,260,270,280,290 or 300%.
25. compositionss according to any one of embodiment 15-24, it comprises any or multiple other
Enzyme.
26. compositionss according to embodiment 25, one or more other enzymes wherein said are selected from following institute
Row：Classify in the xylanase of EC 3.2.1.32, EC 3.2.1.136 or EC 3.2.1.156, cellulase, laminarin
Enzyme, inscribe -1,5- α-L- arabanase, β-D- glucoside glucohydralase, xylobiase, cellobiohydrolase,
Glucosan Isosorbide-5-Nitrae-β-glucosyl enzym, the circumscribed-β of xyloglucan specificity-Isosorbide-5-Nitrae-glucanase and α-N- arabinofuranosidase.
27. enzymes according to embodiment 1-10 or the preparation according to embodiment 14 or according to enforcement
Purposes in producing food, feedstuff or malt beverage product for the compositionss any one of scheme 15-26.
28. enzymes according to embodiment 1-10 or the preparation according to embodiment 14 or according to enforcement
Purposes in producing dough or the roasting product of training for the compositionss any one of scheme 15-26.
29. enzymes according to embodiment 1-10 or the preparation according to embodiment 14 or according to enforcement
Purposes in preparing paper pulp or paper for the compositionss any one of scheme 15-26.
30. enzymes according to embodiment 1-10 or the preparation according to embodiment 14 or according to enforcement
Compositionss any one of scheme 15-26 are used for preparing the purposes of grain component.
31. purposes according to embodiment 29, wherein implementing corn is rye (Secale cereale L.), Semen Tritici aestivi or Fructus Hordei Vulgaris.
32. enzymes according to embodiment 1-10 or the preparation according to embodiment 14 or according to enforcement
Purposes in producing medicated beer or brewage process by-product is modified for the compositionss any one of scheme 15-26.
33. enzymes according to embodiment 1-10 or the preparation according to embodiment 14 or according to enforcement
Purposes in producing wine or fruit juice for the compositionss any one of scheme 15-26.
34. enzymes according to embodiment 1-10 or the preparation according to embodiment 14 or according to enforcement
Use in producing the first generation or second filial generation bio-fuel such as bio-ethanol for the compositionss any one of scheme 15-26
On the way.
A kind of 35. methods of the filterability changing starch-containing material, methods described is included with according to embodiment 1-10
Described enzyme or the preparation according to embodiment 14 or the combination according to any one of embodiment 15-26
The step that thing processes described starch-containing material.
The method of pressure rise during a kind of 36. minimizing filterings in brewageing application, methods described is included with according to reality
Apply the enzyme described in scheme 1-10 or the preparation according to embodiment 14 or according to any one of embodiment 15-26
Described compositions-treated brewages the step with converted mash.
37. one kind are used for producing food, feedstuff or beverage products such as alcoholic beverage or non-alcoholic beverage and are for example based on
The method of the beverage of corn or Fructus Hordei Germinatus such as medicated beer or whiskey, methods described is included with according to embodiment 1-10
Enzyme or the preparation according to embodiment 14 or the compositions-treated according to any one of embodiment 15-26
The step of starch-containing material.
A kind of 38. methods brewageed for production with converted mash, methods described is included with according to embodiment 1-10
Enzyme or the preparation according to embodiment 14 or the compositionss according to any one of embodiment 15-26 at
The step managing starch-containing material.
A kind of 39. methods for producing the first generation or second filial generation bio-fuel such as bio-ethanol, methods described includes
With the enzyme according to embodiment 1-10 or the preparation according to embodiment 14 or according to embodiment 15-26
Any one of the starch-containing material of compositions-treated step.
40. pass through the product that the method according to any one of embodiment 38-39 obtains.
A kind of 41. compositionss, it comprises the product according to embodiment 40, and such as wherein this product is in 0.1%-
In the range of 99.9%.
Embodiment
Embodiment 1
Brewage the related method of xylanase/glucanase and the result of application to application for (filing for).
Following methods have been used for screening the xylanase of application and glucanase in brewageing.
Method：
Water extractible araboxylan (WE-AX) xylanase method：
Sample (to obtain about OD540=0.25-0.30 in this mensure) and xylose standard thing is prepared in distilled water
(0,0.125,0.250,0.375 and 0.500mg/ml distilled water).In time t=0 minute, by 1.75ml solubility Semen Tritici aestivi I
Primary xylan (0.5% Wheat Arabinoxylan (PWAXYH, Megazyme, Irish cloth is auspicious (Bray, Ireland)) in
In 0.1M sodium acetate/acetic acid, pH 5) it is placed in the test tube at 50 DEG C.In t=5 minute, 250 μ l enzymatic solution are added at 50 DEG C
This substrate, then mix.With distilled water as blank.In time t=15 minute, by 2ml DNS solution (1%3,5- dinitros
Base salicylic acid (DNS), 1.6% sodium hydroxide, 30% sodium potassium tartrate tetrahydrate are in distilled water) it is added to enzyme-substrate solution and 2.0ml mark
Quasi- solution.The sample, blank and the reference material that add DNS are placed in 5 minutes in boiling water bath (95 DEG C).Then, by sample, blank
It is placed in 20 minutes in 25 DEG C of water-baths with standard substance and cooled down.Read optical density OD540 of all samples using spectrophotometer.
According to sample dilution, (taking into work) sample size used and standard substance, the xylanase activity of sample can be calculated
Property.
Inscribe-the Isosorbide-5-Nitrae of one unit-beta-xylanase WE-AX activity definition be under these conditions (Water extractible Ah Draw primary xylan (WE-AX) xylanase method) enzyme amount producing 1 micromolar xylose equivalents per minute.
Not extractible araboxylan (WU-AX) the xylanase method of water：
Sample is prepared in distilled water.In time t=0 minute, by insoluble for 1.75ml Wheat Arabinoxylan
(0.5% Wheat Arabinoxylan (PWAXYI, Megazyme, Irish cloth is auspicious (Bray, Ireland)) in 0.1M sodium acetate/
In acetic acid, pH 5) it is placed in the test tube at 50 DEG C.In t=5 minute, 250 μ l enzymatic solution are added to this substrate at 50 DEG C, so
After mix.With distilled water as blank.In time t=15 minute, sample and blank are placed in 5 minutes in boiling water bath (95 DEG C).
Then by sample and blank centrifugation so that the insoluble substrate of remnants precipitates.Using Rouau, X. and Surget, A.
(1994), Carbohydrate Polymers (《Carbohydrate polymer》), 24,123-132 description methods, measure into
Enter the amount of the arabinan in solution.
WU-AX inscribe-Isosorbide-5-Nitrae-beta-xylanase activity definition is the amount (μ g pentose) of the pentose dissolving under these conditions,
Unit definition is μ g pentose/g Xylanase samples.
Xylanase activity algoscopy
Sample (to obtain about OD540=0.25-0.30 in this mensure) and xylose standard thing is prepared in distilled water
(0,0.125,0.250,0.375 and 0.500mg/ml distilled water).In time t=0 minute, by 1.75ml solubility Semen Tritici aestivi I
Primary xylan (0.5% Wheat Arabinoxylan (PWAXYH, Megazyme, Irish cloth is auspicious (Bray, Ireland)) in
In 0.1M sodium acetate/acetic acid, pH 5) it is placed in the test tube at 50 DEG C.In t=5 minute, 250 μ l enzymatic solution are added at 50 DEG C
This substrate, then mix.With distilled water as blank.In time t=15 minute, by 2ml DNS solution (1%3,5- dinitros
Base salicylic acid (DNS), 1.6% sodium hydroxide, 30% sodium potassium tartrate tetrahydrate are in distilled water) it is added to enzyme-substrate solution and 2.0ml mark
Quasi- solution.The sample, blank and the reference material that add DNS are placed in 5 minutes in boiling water bath (95 DEG C).Then, by sample, blank
It is placed in 20 minutes in 25 DEG C of water-baths with standard substance and cooled down.Read optical density OD540 of all samples using spectrophotometer.
According to sample dilution, (taking into work) sample size used and standard substance, the xylanase activity of sample can be calculated
Property.
Inscribe-the Isosorbide-5-Nitrae of one unit-beta-xylanase WE-AX activity definition is that generation 1 per minute is micro- under these conditions
The enzyme amount of mole xylose equivalents.
Dextranase activity algoscopy
Prepare sample (to obtain the OD in standard curve in this mensure in distilled water540) and glucose standard (0,
0.125th, 0.250,0.500 and 0.750mg/ml distilled water).In time t=0 minute, by 1.75ml barley beta-glucan
(1.5% barley beta-glucan (P-BGBM, Megazyme, Irish cloth is auspicious (Bray, Ireland)) is in 1M sodium acetate/acetic acid
In, pH 5) it is placed in the test tube at 50 DEG C.In t=5 minute, 250 μ l enzymatic solution are added to this substrate at 50 DEG C, then mix
Close.With distilled water as blank.In time t=15 minute, by 2ml DNS solution (1%3,5- dinitrosalicylic acids (DNS),
1.6% sodium hydroxide, 30% sodium potassium tartrate tetrahydrate are in distilled water) it is added to enzyme-substrate solution and 2.0ml standard solution.To add
The sample of DNS, blank and reference material are placed in 15 minutes in boiling water bath (95 DEG C).Then, sample, blank and standard substance are placed in
Cooled down within 20 minutes in 25 DEG C of water-baths.Read optical density OD of all samples using spectrophotometer540.According to diluted sample
Degree, (taking into work) sample size used and standard substance, can calculate the dextranase activity of sample.
Inscribe -1 of one unit, 3 (4) -1,4 beta-glucanase activities are defined as (pH 5.0 under conditions of this algoscopy
(or such as specified) and 50 DEG C) enzyme amount producing 1 micromoles glucose equivalent per minute.
Laboratory scale brewage application process：
Pilsen (Pilsner) Fructus Hordei Germinatus using 75: 25-Fructus Hordei Vulgaris are than the water-flour ratio (150ml: 50g flour) with 3: 1
Carry out laboratory scale brewageing applied research.First, batch mixing (mashing in) and pH regulator (5.4,2M H2SO4) it
Before, water is preheating to 53 DEG C.After arriving initial temperature (10 minutes) again, start converted mash preparation process (referring to Fig. 1) and
Add enzyme.After mashing off (mashing off), using conventional plastic funnel and filter paper, (Britain irrigates for No. 1 filter paper, diameter 24cm
Te Man (Whatman, England)) carry out beerwort separation.Assessment strainability and several others beerwort parameter, than
As, i.e. viscosity, beta glucan and pentosan.
Wort filtration is measured 30 minutes, then terminate to filter.Before carrying out any further analysis, will
The beerwort cooling collected.
Filter
Cross filter data to collect for blank (being added without exogenous enzyme when brewageing) after 5,10,15 and 30 minutes
Wort volume represents.
Pilot-scale is brewageed
Brewage in facility (2HL capacity) in pilot-scale and tested.Carry out beerwort by filtering to separate, and pass through water
Flat kieselguhr filters and carries out beer filtration.
For being illustrated in the combination of glucanase and xylanase under the conditions of " challenging " brewages to the optimization function filtering,
Carry out pilot-scale wine-making using the mixing flour comprising 75% Fructus Hordei Germinatus and 25% Fructus Hordei Vulgaris.First, by water：Flour is than setting
For 2.8: 1 (converted mash preparation starts), increase to 3.1: 1 when filtering starts.By contrast, in the filtering of commercial scale brewery
In, water: flour is than generally in 3.2-3.8.Therefore, current pilot plant test is set as 3.1: 1 water: flour ratio is it is believed that be partial to
Challenging.
Using two-roller mill machine dry grinding Fructus Hordei Germinatus and Fructus Hordei Vulgaris.Fructus Hordei Vulgaris and Fructus Hordei Germinatus all use the roll spacing of about 0.7mm to grind twice.
Batch mixing is carried out with 53 DEG C of initial mashing wine with dregs temperature.After batch mixing, carry out following little adjustment：Adjustment converted mash body
The long-pending water that makes: flour ratio is for 2.8: 1, and pH is adjusted to about 5.56 (lactic acid).After having finely tuned converted mash, add enzyme, then
Converted mash preparation process shown in execution Fig. 1.Saccharifying at 70 DEG C stopped follow procedure for 15 minutes, but will prolong the stop time
Long 5 minutes, until iodine test shows that no starch exists.(Ludwig Narziss and Werner Back, Technical University at Munich
(brewage and be, Wei Sen brewery (Weihenstephan)),《Brewing factory is summarized》(Abriss der Bierbrauerei),
WILEY-VCH Verlags GmbH publishing company, German Wei Yinhaimu (Weinheim), 2005).
The mashing off at 78 DEG C is proceeded by after stopping 5 minutes.Converted mash is transferred to filter tank, this groove is pre-loaded into water in advance
To the just height below at " false bottom ".Converted mash standing is allowed so that filter cake is precipitated.Then carry out the circulation of 15 minutes
(140L/h), to guarantee filter cake precipitation and beerwort clarification.Generally in plant-scale brewageing, filter by reach given
During beerwort turbidity, but in this test, circulation is held constant at 15 minutes, so as to carrying out to each test
Relatively.Collection data below during filtering, the wort volume (L) including the time (min), collected, the filtration pressure of filter cake
Difference (mmWC, mm water column), pump discharge (%), beerwort turbidity (EBC) and converted mash temperature (DEG C).
In filter process, the pressure rise of filter cake is presently considered to be one and contributes to setting the standard of beerwort filtration properties
Factor.Reach for example, 250mmWC of very high pressure reduction-during first wort is collected and during remaining filtering
For example, 450mmWC- then causes filter cake to destroy (racking) (also referred to as deep (deep cut)).It is such a that filter cake destroys
Process, i.e. filter cake collapse, or by cutting the formation of filter cake removal filtration channel slowly with specially designed knife.Destroy in filter cake
Afterwards, introduce the beerwort circulation (flow velocity of 6 minutes：120l/h) so that filter cake is ready to proceed to filter.Filter cake destroys and makes
Originally the strainability that can be weakened is released, if to be also resulted in malt juice quality by weakening poor for strainability.If to the 3rd
Secondary drip washing is also not introduced into, till starting, the destruction that pressure is led to, then automatically broken when the 3rd time and the 4th drip washing start
Bad, to guarantee just complete before beerwort separates to be not in completely to filter to block.
Carry out filtering with the setting shown in table 1.
Table 1：Filtering is arranged.Volume (L), filtration flow-rate (L/h) and the elution volume (L) collected.
| Beerwort | The volume collected, L | Filtration flow-rate, L/h | Elution volume, L |
| First wort | 0-60 | 130 | |
| 1st drip washing | 60-78 | 140 | 18 |
| 2nd drip washing | 78-96 | 160 | 18 |
| 3rd drip washing | 96-114 | 180 | 18 |
| The 4th drip washing | 114-140 | 180 | 26 |
After filtering terminates, sweet beerwort is returned converted mash groove, be heated to boiling, add Flos lupuli (Flos Humuli Lupuli).Continuously add Flos lupuli (Flos Humuli Lupuli) 80
Minute, terminate when adding Flos lupuli (Flos Humuli Lupuli) to adjust pH to 5.10 ± 0.05.Using whirlpool, Flos lupuli (Flos Humuli Lupuli) is removed from bitter beerwort, then
Beerwort is cooled to about 8 DEG C.For being fermented, select submerged fermentation dry yeast (the wine brewing ferment from Fermentis company
Female (Saccharomyces cerevisiae)) W34/70.By yeast rehydration 30 minutes, inoculated with 100g/HL.Main fermentation
Keep 5-6 days at 10 DEG C, then in 15 DEG C of maturations, until disappear sugar and biacetyl are in below 80ppb.By medicated beer at 1 DEG C and 0.7
Refilter after other 2-3 week of preservation under bar.
Using 1.2 μm of PP candle plates and kieselguhr, medicated beer is carried out horizontal filtration.8 at most can be put in filter element
Block plate, obtains total filter area of about 0.5m2.Put 3 blocks of plates in our current research, filtered under the flow velocity of 130L/h, obtain
The rate of filtration to 6.9HL/ (h m2).In plant-scale brewery, the rate of filtration is generally set to 5-7HL/ (h
M2 between).As can be seen here, current be set at high-end-this is to be intentionally chosen it is intended to challenge beer filtration condition, to test
Card selects the potential benefit brought using enzyme in brewage process.During beer filtration, monitor flow velocity (L/h) and pressure
Force value (inlet pressure and outlet pressure) is to verify beer filtration performance.Also carried out multiple beer analysis, such as original content (OG),
Apparent extract (AE), alcohol by volume (ABV), apparent attenuation (ADF), real degree of attenuation (RDF), pH, colourity and bitterness, with
Assessment beer quality.
Result：
Xylanase：
For xylanase, the activity of soluble substrate and insoluble substrate, the pH of xylanase and temperature characterisitic are screened
Xylanase.
Result is displayed in Table 2.
The xylanase that table 2. is screened, they are to solubility (WE-AX) and insoluble (WU-AX) araboxylan bottom The activity of thing and their biochemical characteristics in terms of temperature and pH.
WE-AX and WU-AX enzymatic activity (U) as "Water extractible araboxylan (WE-AX) xylanase method " and Measure described in " not extractible araboxylan (WU-AX) the xylanase method of water " chapters and sections.
According to the result of biochemical screening, select the activity to solubility araboxylan and to insoluble Arabic wood
The suitable xylanase of the ratio of the activity of polysaccharide, for further being tested in application test.Result shows in table 3
Show.
Table 3：The xylanase that screened and using xylanase contrast blank (no xylanase) obtained relative Extractum yield.Finally, with xylanase to the activity of insoluble araboxylan and to solubility araboxylan The ratio (WU-AX/WE-AX) of activity represents xylanase substrate specificity.
By above description (" filtration ") measure strainability, and with different time points with respect to negative control (blank)
Filtrate volume represent.
WE-AX and WU-AX enzymatic activity (U) as "Water extractible araboxylan (WE-AX) xylanase method " and Measure described in " not extractible araboxylan (WU-AX) the xylanase method of water " chapters and sections.
Glucanase：
Activity for glucanase and temperature characterisitic screening glucanase, result is displayed in Table 4.
Table 4：The glucanase being screened, their activity and the biochemical characteristic in terms of temperature
N.d.=undetermined
It is measured described in dextranase activity/unit dextranase activity described above algoscopy.
According to the result of biochemical screening, select there is the glucanase of suitable characteristic for entering to advance in application test
The test of one step.Result is displayed in Table 5.
Table 5：The title of the glucanase being screened and source and obtained using glucanase contrast blank (no enzyme) Relative extractum yield.
By above description (" filtration ") measure strainability, and with different time points with respect to negative control (blank)
Filtrate volume represent.
According to the independent screening to xylanase and glucanase, it is combined testing, result is displayed in Table 6.
Table 6：Xylanase and glucanase contrast blank and contrast Wine obtained by the group practicess of Max Make application result.250 fungal xylanases units FXU-S/g, 700 Cellulase Unit EGU/g (Novozymes company of Denmark) Result is represented with the relative extractum yield being obtained.
(The source of Max may include other microorganisms beyond microorganism Aspergillus aculeatus (A.aculeatus), such as
Described in WO05059084)
By above description (" filtration ") measure strainability, and with different time points with respect to negative control (blank)
Filtrate volume represent.
The facility of 2HL pilot-scale is further tested to suitable combination to verify, result is in table 7 He
Show in Fig. 3.
Table 7：Pilot-scale obtained by the checking of glucanase and xylanase screening brewages application result.Contrast is blank With Max tests bacillus subtilises (B.sub) glucanase S and Tabin aspergillus (A.tub) xylanase Combination.Collected data is that the gross pressure during mean flow rate (L/h), filtering raises record during (mm WC) and filtering Maximum pressure (mm WC).
Embodiment 2
In the present embodiment it is intended to confirm that the xylanase for brewageing application can be Arabic to high-molecular-weight soluble wooden
Polysaccharide (HMWS-AX) and the extractible araboxylan of water (WE-AX) have very high selectivity.It is believed that thus only having
The araboxylan of limit quantity needs to be dissolved.Therefore, related abnormal flavour Potential feasibility greatly reduces.
Significantly reduced viscosity is conducive to converted mash to separate with medicated beer.For brewageing application, preferable xylanase is special
Property may include one of each side of table 8 below or many aspects：
As shown in Figure 3, the water-insoluble araboxylan (WU-AX) in corn and the filter cake stability in brewery
Relevant.
Ferulic acid (FA) concentration in corn is highly dependent upon tissue.Find highest concentration in peel material, and
Concentration in endosperm is much lower.Report different concentration.2700 μ g/g insoluble fibres, 185 μ g/g soluble fiber dense
Degree be possible (Bunzel et al., 2001, Journal of Sc.of food and agriculture (《Food and agricultural
Magazine》), volume 81, the 653-60 page).
Intuitively, it means every 200 xylose molecules in the araboxylan in insoluble fibre (WU-AX)
Only find a FA, and be then that every 2500 xyloses only find a FA in soluble fiber (WE-AX).
It is a well known fact that xylanase can lead to abnormal flavour to be formed in medicated beer, such as dissociate ferulic acid and 4-VG.
Method：
According to the standard mentioned in table 8+9 it was found that 15 kinds derive from Du Pont industrial bio scientific company (DuPont
Industrial Biosciences) xylanase be potential material standed for.These xylanase are in laboratory converted mash system
Combine with Fructus Hordei Germinatus and screen using most 30% Fructus Hordei Vulgaris in standby application.Converted mash separating rate, pentosan/Arab are monitored
Xylan level and Wort viscosity etc..Test optimal several material standed fors in the research of Ji Xiang pilot scale brewery, to check
Xylanase characteristic is simultaneously associated by our hypothesis with the feature foundation in brewageing.By select xylanase material standed for
1,4 beta-glucanase is combined, the optimal dose of the xylanase that test is selected.
Result and discussion：
It is unique variable that pilot plant brewages middle enzyme dosage.Applying WU-AX selectivity xylanase (X1) leads to filter bed to collapse.
WE-AX selectivity xylanase material standed for (reference substance, X2, X3) leads to low pressure rise.Reference substance is xylanase+β-Portugal
The blend of dextranase.
Table 11：Beerwort analysis-pilot plant is studied
| Sample number into spectrum | Reference substance | X+B | Xh+B |
| Extractum (° Plato) | 15.70 | 16.00 | 15.95 |
| Beta glucan (mg/1) in beerwort | 44 | 35 | 25 |
| Dynamic viscosity (mPa.s) under 12 ° of Plato | 1.65 | 1.68 | 1.68 |
| Total pentosan (mg/l) | 3540 | 2970 | 3010 |
Table 12：Strake (Strecker) the aldehyde analysis of ageing medicated beer
The WE-AX selectivity xylanase being applied with median dose (X) and high dose (Xh) and 1,4 beta-glucanase (B) mutually group
The optimization blend closing, acts on 20% Fructus Hordei Vulgaris/80% Fructus Hordei Germinatus.Result shows that converted mash and medicated beer separating property are good, abnormal flavour
Formed and the risk of filter bed collapse is low.
Conclusion
This research demonstrates to apply to have brewageing of high selectivity to WE-AX in converted mash preparation process uses xylan
The importance of enzyme.Obtain following beneficial effect：
Converted mash separates and beer filtration is functional
During filtering, the risk of filter bed collapse reduces to minimum
Decompose the Potential feasibility attenuating that related abnormal flavour is formed to araboxylan
Tolerance xylanase is excessive
Xylanase usually can combine applying with 1,4 beta-glucanase, and beneficial effect substantially, is easy to control and was separated
Journey.
Embodiment 3：
Assessment X3/BglS (also referred to as AtuXyn3/BsuGluS) combination in 2HL pilot scale wine-making
Materials and methods：
Experiment：Enzyme：
AtuXyn3(X3)/BsuGluS(Bgls)(a)：BglS (bacillus cereuss glucanase) and X3 (aspergillosiss xylan
Enzyme：BgLS：0.50mg albumen/kg flour, X3：1.50mg albumen/kg flour) combination.
AtuXyn3(X3)/BsuGluS(Bgls)(b)：As AtuXyn3 (X3)/BsuGluS (Bgls) (a), but X3 dosage
Increase by 20% to test steadiness (robustness).
Reference substance：Benchmark enzyme product (Max), dosage is 0.20kg/T flour.
Raw material：
Adjuvant：Fructus Hordei Vulgaris 22% w/w.
Fructus Hordei Germinatus：Pilsen (Pilsner) Fructus Hordei Germinatus Chiraz 42.6% w/w, Pilsen Fructus Hordei Germinatus Quench DMG
35.4% w/w (w/w).
The material that all acid for pH, calcium, zinc and bitterness levels are adjusted is food stage, and is considered as the wine of standard
Producing material material.
Brewage the medicated beer style that formula is intended to brew international lager beer.
Pulverizing：
K ü nzel 2 roller pilot scale is ground.The material grinding simulates 4 roller mills twice by roller.
Fructus Hordei Germinatus flour：Mill running, passing through for the first time during roller is 1.5mm, passes through for second during roller to be 0.7mm.
Fructus Hordei Vulgaris flour：Mill running, passing through for the first time during roller is 1.5mm, passes through for second during roller to be 0.4mm.
Brewery 2HL：
All brewage be based on 190L beerwort HGB (high concentration is brewageed) leach converted mash preparation and standard filtering, mesh
Mark is 16 ° of Plato.During the filtering being carried out with fixed flow rate, record differential pressure (parameters as assessment filtration properties).Institute
Brewage material all to do sth. in advance (24h) grinding and be maintained at before contacting with water in sealing bucket.All material is all in converted mash system
Put in converted mash preparation tank in after standby beginning first 3 minutes.Carried out calcium and pH adjustment before adding enzyme.PH (20 DEG C) is 52
DEG C interrupt when reexamined.Confirm iodine equivalent concentration at 72 DEG C after 10 minutes.Carry out filtering at 78 DEG C.
During first wort is collected, filtration properties are assessed with the fixed flow rate of 90l/H.Receive in drip washing and dilute beerwort
During collection, flow velocity is brought up to 110L/ hour and 130L/ hour.Chemical analyses are carried out to cold beerwort.
Beerwort boils：
Boiled for 4-5% external boiler using evaporation rate.Add hop extract, target when beginning beerwort boils
It is that final medicated beer reaches 20BU.
Fermentation 50L：
All fermentations are all carried out in 50L cone tank.Fermented according to the operation sequence of standard.With 15 × 106Individual
Active yeast cell/ml is inoculated.Carry out yeast counts using Nucleo enumerator and calculate survival rate.
Medicated beer is processed：
With constant pressure operation flame filter press.Flow velocity assessment is carried out by weight.
Collect data from 1 and 3 screen plates.
De- wine (debrewing)：
All medicated beer carry out de- wine to 5.0%ABV alcohol by volume, and this is considered as international lager beer standard.
Bottling：
By CO2Adjust to 5.0g/L.All beer samples evacuate dress using single all on McLennon automatic filling machine
Enter in 33cl standard jar.
Beer analysis：
Analyze young beer using GC-MS.
Measure chemical ageing process using GC-MS.
Result and observation：Prepared by converted mash.
Carry out converted mash preparation with following condition：
Simulate within 10 minutes the 15-20 minute converted mash preparation that service firing grinding machine (running mill) is carried out for 52 DEG C.
65 DEG C 40 minutes.
72 DEG C 30 minutes.
78 DEG C 10 minutes.
All heating steps are all executed with 1 DEG C.Fig. 7 provides schematic diagram.
All tests are all carried out with this converted mash preparation scheme, and target is that 16 ° of Plato brewage.To this processing step
Do not comment on.
Result and observation：Filtering.
Carry out filtering in 2hl brewery, useful load is 150kg/m2.This represents brewery's operation of standard.Control filter
Clear process is in 100 ls/h of fixed flow rate average out to.Initial flow rate is 90 ls/h, improves during dilute beerwort is collected
To 130 ls/h.Four are brewageed and have recorded differential pressure and online turbidimetry.Carry out whole filterings and wheat in about 2 hours
Bud juice is collected.
X3/BglS (b) and X3/BglS (a) test is asserted the test being have optimal filtration properties, next to that X3/
BglS (a) tests, and the performance of UF max test is worst.
Table 13：The data collected during the converted mash filtering of four tests.
| UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) | |
| Filter tank useful load (kg/m3) | 153 | 153 | 153 | 153 |
| The filter tank time (minute) | 154 | 164 | 170 | 154 |
| Differential pressure (cm) | 40 | 30 | 30 | 30 |
| Destroy (# is deep) | 1 | 1 | 1 | 1 |
| Turbidity (EBC) | 10 | 15 | 10 | 10 |
| First wort pressure rise (cm/h) | 40 | 33 | 31 | 30 |
| The deep time (minute) for the first time | 45 | 60 | 120 | 115 |
" differential pressure " of standard and " first wort pressure rise " is measured with cmWC (cm water column), rather than respectively with (cm) and
(cm/h) measure.
Result and observation：Beerwort analysis after boiling.
The analysis of cold beerwort shows similar result.Beta glucan analysis shows sample room slightly difference.
Table 14：The chemical analyses of cold beerwort.
| Beerwort | UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) |
| Extractum (%plato) | 16.09 | 16.05 | 15.99 | 16.1 |
| Colourity (EBC) | 9.7 | 9.3 | 9.3 | 9.3 |
| pH | 5 | 5.2 | 5.2 | 5.2 |
| Iodine (Y/N) | N | N | N | N |
| Bitterness (BU, EBC) | 52 | 51 | 46 | 50 |
Table 15：The analytical data of cold beerwort.
| UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) | |
| Beta glucan (mg/L) in beerwort | 49 | 40 | 25 | 30 |
| Dynamic viscosity (mPa.s) at 12 DEG C | 1,888 | 1,685 | 1,679 | 1,686 |
| Pentosan (mg/L) | 3365 | 2975 | 3014 | 2964 |
| Ferulic acid (ug/mL) | 4,3 | 3,9 | 3,8 | 3,9 |
| 4-VG(ug/ml) | ＜ 0,49 | ＜ 0,49 | ＜ 0,49 | ＜ 0,49 |
(12 DEG C is 12 ° of Plato)；(%Plato can be with a ° Plato used interchangeably)
Result and observation：Fermentation.
The analysis of unpasteurized beer is given in table 16.
Table 16：Unpasteurized beer is analyzed.
| Unpasteurized beer | UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) |
| Ethanol (%vol) | 6.79 | 6.79 | 6.7 | 6.86 |
| True extractum (%P) | 6.28 | 6 | 6 | 6 |
| RDF (%) | 63.5 | 63.7 | 63.5 | 64.4 |
| Original extractum (%P) | 16.29 | 16.25 | 16.09 | 16.24 |
| Colourity (EBC) | 8.3 | 8.5 | - | - |
| pH | 4.4 | 4.4 | 4.4 | 4.4 |
| S02(ppm) | 7 | 9 | 11 | 10 |
| Bitterness (BU, EBC) | 29 | 28 | 27 | 27 |
There is between each test of unpasteurized beer analysis display the similarity of height.All tests are respectively provided with relatively low RDF
(real degree of attenuation), but this typically by weight/weight (w/w) calculates and adds 22% Fructus Hordei Vulgaris to be seen.
Result and observation：Beer filtration.
Beer sample is filtered with fixation pressure using flame filter press.Two kegs of about 15kg are filtered, often
The filter data of crossing of individual keg is given in table 17.First keg is filtered with 1 filter disc, and the second keg is filtered with 3 filter discs.Differential pressure
Always 0.5 bar.Screen plate is the KD7 (20cm × 20cm) from Begerow company.
The filtration curve overview image of 1 filter plate or 3 filter plate is identical.It is considered that 1
Screen plate record may be excessively sensitive and can not show that real ratio is poor.
Table 17：Filter data crossed by the keg of four tests
| Filter | UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) |
| - 1 filter disc of the rate of filtration (L/h) | 4.8 | 5.6 | 9.9 | 11.8 |
| - 3 filter discs of the rate of filtration (L/h) | 77.2 | 59.6 | 70.6 | 105.4 |
Result and observation：Final beer analysis.
Test medicated beer is analyzed and is displayed in Table 18 according to the operation sequence (EBC) of standard.
Table 18：Final beer analysis.
| Finished beer | UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) |
| Ethanol (%) | 4.82 | 4.89 | 5.01 | 4.92 |
| True extractum (%P) | 4.5 | 4.6 | 4.6 | 4.4 |
| RDF (%) | 63.2 | 63.4 | 63.8 | 64.3 |
| Original extractum (%P) | 11.85 | 11.99 | 12.19 | 11.89 |
| Colourity (EBC) | 4.8 | 4.9 | 5 | 5 |
| pH | 4.4 | 4.4 | 4.4 | 4.4 |
| S02(ppm) | 13 | 13 | 10 | 6 |
| Bitterness (BU, EBC) | 22 | 22 | 20 | 18 |
| Turbidity (EBC) | 0.43 | 0.4 | 0.38 | 0.4 |
| - 60 DEG C of total turbidity -5 days (EBC) | 8.6 | 12.9 | 7.3 | 6.2 |
| C02(g/L) | 4.9 | 5.3 | 5.1 | 5.2 |
| Biacetyl (ppb) | 12 | 11 | 8 | 10 |
| Head retention (S) | 107 | 111 | 119 | 108 |
| Foam volume (mL) | 452 | 476 | 460 | 470 |
Result and observation：Strecker aldehyde in final medicated beer and " ageing mark ".
Unpasteurized beer and ageing medicated beer are all analyzed.By GC-MS to unpasteurized beer and ageing beer analysis
Strecker aldehyde and " ageing and hot mark " (2-Me-Pr (2 methyl propanal), 2-Me-Bu (2 methyl butyraldehyde), 3-Me-Bu
(3- methylbutyraldehyd), furfural, 3- methylthiopropionaldehyde, PheAcal (ethylalbenzene) and T2N (trans -2- nonenyl aldehyde)).Unpasteurized beer
Analytical data is given in table 19.
Table 19：The Strecker aldehyde analysis of unpasteurized beer.Heat and ageing mark (furfural and trans -2- nonenyl aldehyde) are used as sample Product compare.
| Ageing mark (unpasteurized beer) | UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) |
| 2-ME-Pr(ppb) | 5 | 5 | 5 | 6 |
| 2-ME-Bu(ppb) | 2 | 2 | 2 | 2 |
| 3-ME-Bu(ppb) | 6 | 6 | 6 | 6 |
| Furfural (ppb) | 10 | 11 | 11 | 10 |
| 3- methylthiopropionaldehyde (ppb) | 4 | 4 | 4 | 4 |
| PheAcal(ppb) | 6 | 6 | 6 | 7 |
| T2N(ppb) | 0.0011 | 0.005 | 0.004 | 0.006 |
Test medicated beer is incubated after 2 week at 37 DEG C and carries out Strecker aldehyde analysis.The data of ageing beer sample
Table 20 is given.
Table 20：The Strecker aldehyde analysis of ageing medicated beer.Heat and ageing mark (furfural and trans -2- nonenyl aldehyde) are used as Sample controls.
| Ageing mark (forces ageing medicated beer) | UF max | X3/BglS(a) | X3/BglS(b) | X3/BglS(a) |
| 2-ME-Pr(ppb) | 25 | 23 | 22 | 26 |
| 2-ME-Bu(ppb) | 3 | 3 | 3 | 2 |
| 3-ME-Bu(ppb) | 9 | 7 | 7 | 8 |
| Furfural (ppb) | 111 | 92 | 93 | 78 |
| 3- methylthiopropionaldehyde (ppb) | 6 | 5 | 6 | 6 |
| PheAcal(ppb) | 10 | 9 | 9 | 10 |
| T2N(ppb) | 0.017 | 0.022 | 0.022 | 0.022 |
The data display being given in table 20 goes out Strecker aldehyde level and improves as expected.Furfural and trans -2- nonyl
The raising of olefine aldehydr reaches expected level.
Conclusion：
According to the experiment of pilot-scale, we it could be assumed that, in this experiment, the ratio of BglS and X3 of test shows
Reference substance UltraFlo Max in brewageing than pilot-scale is equally good or even better.
By using challenge raw material add 22% Fructus Hordei Vulgaris and combine with the Fructus Hordei Germinatus containing 300mg/l beta glucan
See, result is unexpected.This performance is not only seen in converted mash separating resulting, also sees in beer filtration.Due to
When using BREW2 (pentosan data), the dissolubility of cell wall material is low, recordable to being likely to result in and abnormal flavour and stable
Property the related cell wall material of quality problems degree relatively low.
Last it could be assumed that, the dosage of the xylanase composition in X3/Bgls (b) improves 20% and seems not to any institute
The parameter of assessment has any impact, and this shows that X3/Bgls (a) is a firm enzyme combination.
Sequence：
AtuXyn3, Tabin aspergillus (Aspergillus tubigensis) (SEQ ID NO：1), 302 aminoacid
QASVSIDTKFKAHGKKYLGNIGDQYTLTKNSKTPAIIKADFGALTPENSMKWDATEPSRGQFSFSGSDY
LVNFAQSNNKLIRGHTLVWHSQLPSWVQAITDKNTLIEVMKNHITTVMQHYKGKIYAWDVVNEIFNEDGSLRDSVFY
QVIGEDYVRIAFETARAADPNAKLYINDYNLDSASYPKLTGMVSHVKKWIEAGIPIDGIGSQTHLSAGGGAGISGAL
NALAGAGTKEIAVTELDIAGASSTDYVEVVEACLDQPKCIGITVWGVADPDSWRSSSTPLLFDSNYNPKPAYTAIAN
AL
TerXyn1, Ai Mosen wart penicillium sp (Talaromyces emersonii) (Geosmithia emersonii (Taleromyces
emersonii))(SEQ ID NO：2)
AGLNTAAKAIGLKYFGTATDNPELSDTAYETQLNNTQDFGQLTPANSMKWDATEPEQNVFTFSAGDQIA
NLAKANGQMLRCHNLVWYNQLPSWVTSGSWTNETLLAAMKNHITNVVTHYKGQCYAWDVVNEALNDDGTYRSNVFYQ
YIGEAYIPIAFATAAAADPNAKLYYNDYNIEYPGAKATAAQNLVKLVQSYGARIDGVGLQSHFIVGETPSTSSQQQN
MAAFTALGVEVAITELDIRMQLPETEALLTQQATDYQSTVQACANTKGCVGITVWDWTDKYSWVPSTFSGYGDACPW
DANYQKKPAYEGILTGLGQTVTSTTYIISPTTSVGTGTTTSSGGSGGTTGVAQHWEQCGGLGWTGPTVCASGYTCTV
INEYYSQCL
AtuXyn4, Tabin aspergillus (Aspergillus tubigensis) (SEQ ID NO：3)
EPIEPRQASVSIDTKFKAHGKKYLGNIGDQYTLTKNSKTPAIIKADFGALTPENSMKWDATEPSRGQFS
FSGSDYLVNFAQSNNKLIRGHTLVWHSQLPSWVQSITDKNTLIEVMKNHITTVMQHYKGKIYAWDVVNEIFNEDGSL
RDSVFYKVIGEDYVRIAFETARAADPNAKLYINDYNLDSASYPKLTGMVSHVKKWIAAGIPIDGIGSQTHLSAGGGA
GISGALNALAGAGTKEIAVTELDIAGASSTDYVEVVEACLNQPKCIGITVWGVADPDSWRSSSTPLLFDSNYNPKPA
YTAIANAL
AacXyn2, microorganism Aspergillus aculeatus (Aspergillus aculeatus) (SEQ ID NO：4)
MVGLLSITAALAATVLPNIVSAVGLDQAAVAKGLQYFGTATDNPELTDIPYVTQLNNTADFGQITPGNS
MKWDATEPSQGTFTFTKGDVIADLAEGNGQYLRCHTLVWYNQLPSWVTSGTWTNATLTAALKNHITNVVSHYKGKCL
HWDVVNEALNDDGTYRTNIFYTTIGEAYIPIAFAAAAAADPDAKLFYNDYNLEYGGAKAASARAIVQLVKNAGAKID
GVGLQAHFSVGTVPSTSSLVSVLQSFTALGVEVAYTEADVRILLPTTATTLAQQSSDFQALVQSCVQTTGCVGFTIW
DWTDKYSWVPSTFSGYGAALPWDENLVKKPAYNGLLAGMGVTVTTTTTTTTATATGKTTTTTTGATSTGTTAAHWGQ
CGGLNWSGPTACATGYTCTYVNDYYSQCL
TreXyn3, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：5)
MKANVILCLLAPLVAALPTETIHLDPELAALRANLTERTADLWDRQASQSIDQLIKRKGKLYFGTATDR
GLLQREKNAAIIQADLGQVTPENSMKWQSLENNQGQLNWGDADYLVNFAQQNGKSIRGHTLIWHSQLPAWVNNINNA
DTLRQVIRTHVSTVVGRYKGKIRAWDVVNEIFNEDGTLRSSVFSRLLGEEFVSIAFRAARDADPSARLYINDYNLDR
ANYGKVNGLKTYVSKWISQGVPIDGIGSQSHLSGGGGSGTLGALQQLATVPVTELAITELDIQGAPTTDYTQVVQAC
LSVSKCVGITVWGISDKDSWRASTNPLLFDANFNPKPAYNSIVGILQ
TreXyn5, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：6)
QCIQPGTGYNNGYFYSYWNDGHGGVTYCNGPGGQFSVNWSNSGNFVGGKGWQPGTKNRVINFSGSYNPN
GNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQRVNQPSIIGTATFYQYWSVRRNHRS
SGSVNTANHFNAWAQQGLTLGTMDYQIVAVEGYFSSGSASITVSD
BsuGluS, bacillus subtilises (Bacillus subtilis) (SEQ ID NO：7), 214 aminoacid
QTGGSFFDPFNGYNSGFWQKADGYSNGNMFNCTWRANNVSMTSLGEMRLALTSPAYNKFDCGENRSVQT
YGYGLYEVRMKPAKNTGIVSSFFTYTGPTDGTPWDEIDIEFLGKDTTKVQFNYYTNGAGNHEKIVDLGFDAANAYHT
YAFDWQPNSIKWYVDGQLKHTATNQIPTTPGKIMMNLWNGTGVDEWLGSYNGVNPLYAHYDWVRYTKK
TerGlu1, Ai Mosen wart penicillium sp (Talaromyces emersonii) (Geosmithia emersonii (Taleromyces
emersonii))(SEQ ID NO：8)
APVKEKGIKKRASPFQWFGSNESGAEFGNNNIPGVEGTDYTFPNTSAIQILIDQGMNIFRVPFLMERMV
PNQMTGPVDSAYFQGYSQVINYITSHGASAVIDPHNFGRYYNNIISSPSDFQTFWHTIASNFADNDNVIFDTNNEYH
DMDESLVVQLNQAAIDGIRAAGATSQYIFVEGNSWTGAWTWTQVNDAMANLTDPQNKIVYEMHQYLDSDGSGTSDQC
VNSTIGQDRVESATAWLKQNGKKAILGEYAGGANSVCETAVTGMLDYLANNTDVWTGAIWWAAGPWWGDYIFSMEPP
SGIAYEQVLPLLQPYL
BsuGlu103FULL, bacillus subtilises (Bacillus subtilis) (SEQ ID NO：9)
DDYSVVEEHGQLSISNGELVNERGEQVQLKGMSSHGLQWYGQFVNYESMKWLRDDWGITVFRAAMYTSS
GGYIDDPSVKEKVKETVEAAIDLGIYVIIDWHILSDNDPNIYKEEAKDFFDEMSELYGDYPNVIYEIANEPNGSDVT
WDNQIKPYAEEVIPVIRDNDPNNIVIVGTGTWSQDVHHAADNQLADPNVMYAFHFYAGTHGQNLRDQVDYALDQGAA
IFVSEWGTSAATGDGGVFLDEAQVWIDFMDERNLSWANWSLTHKDESSAALMPGANPTGGWTEAELSPSGTFVREKI
RESASIPPSDPTPPSDPGEPDPGEPDPTPPSDPGEYPAWDSNQIYTNEIVYHNGQLWQAKWWTQNQEPGDPYGPWEP
LKSDPDSGEPDPTPPSDPGEYPAWDSNQIYTNEIVYHNGQLWQAKWWTQNQEPGDPYGPWEPLN
TreGlu2, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：10)
QQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPPTSSGVRFAGV
NIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNDDGMTIFRLPVGWQYLVNNNLGGNLDSTSISK
YDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQLAS
KYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGST
TNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYL
GYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK
TreGlu3, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：11)
QTSCDQWATFTGNGYTVSNNLWGASAGSGFGCVTAVSLSGGASWHADWQWSGGQNNVKSYQNSQIAIPQ
KRTVNSISSMPTTASWSYSGSNIRANVAYDLFTAANPNHVTYSGDYELMIWLGKYGDIGPIGSSQGTVNVGGQSWTL
YYGYNGAMQVYSFVAQTNTTNYSGDVKNFFNYLRDNKGYNAAGQYVLSYQFGTEPFTGSGTLNVASWTASIN
TreGlu4, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：12)
HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGFVSPDAYQNPDIICHKNATNAKGHASV
KAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPD
NLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPT
VVSGLPTSVAQGSSAATATASATVPGGGSGPTSRTTTTARTTQASSRPSSTPPATTSAPAGGPTQTLYGQCGGSGYS
GPTRCAPPATCSTNPYYAQCLN
TreGlu6, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：13)
AFSWKNVKLGGGGGFVPGIIFHPKTKGVAYARTDIGGLYRLNADDSWTAVTDGIADNAGWHNWGIDAVALDPQDDQK
VYAAVGMYTNSWDPSNGAIIRSSDRGATWSFTNLPFKVGGNMPGRGAGERLAVDPANSNIIYFGARSGNGLWKSTDG
GVTFSKVSSFTATGTYIPDPSDSNGYNSDKQGLMWVTFDSTSSTTGGATSRIFVGTADNITASVYVSTNAGSTWSAV
PGQPGKYFPHKAKLQPAEKALYLTYSWWPDAQLFRSTDSGTTWSPIWAWASYPTETYYYSISTPKAPWIKNNFIDVT
SESPSDGLIKRLGWMIESLEIDPTDSNHWLYGTGMTIFGGHDLTNWDTRHNVSIQSLADGIEEFSVQDLASAPGGSE
LLAAVGDDNGFTFASRNDLGTSPQTVWATPTWATSTSVDYAGNSVKSVVRVGNTAGTQQVAISSDGGATWSIDYAAD
TSMNGGTVAYSADGDTILWSTASSGVQRSQFQGSFASVSSLPAGAVIASDKKTNSVFYAGSGSTFYVSKDTGS
SFTRGPKLGSAGTIRDIAAHPTTAGTLYVSTDVGIFRSTDSGTTFGQVSTALTNTYQIALGVGSGSNWNLYAFGTGP
SGARLYASGDSGASWTDIQGSQGFGSIDSTKVAGSGSTAGQVYVGTNGRGVFYAQGTVGGGTGGTSSSTKQSSSSTS
SASSSTTLRSSVVSTTRASTVTSSRTSSAAGPTGSGVAGHYAQCGGIGWTGPTQCVAPYVCQKQNDYYYQCV
TreGlu7, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：14)
HGQVQNFTINGQYNQGFILDYYYQKQNTGHFPNVAGWYAEDLDLGFISPDQYTTPDIVCHKNAAPGAIS
ATAAAGSNIVFQWGPGVWPHPYGPIVTYVVECSGSCTTVNKNNLRWVKIQEAGINYNTQVWAQQDLINQGNKWTVKI
PSSLRPGNYVFRHELLAAHGASSANGMQNYPQCVNIAVTGSGTKALPAGTPATQLYKPTDPGILFNPYTTITSYTIP
GPALWQG
TreGlu8, trichoderma reesei (Trichoderma reesei) (SEQ ID NO：15)
GKIKYLGVAIPGIDFGCDIDGSCPTDTSSVPLLSYKGGDGAGQMKHFAEDDGLNVFRISATWQFVLNNT
VDGKLDELNWGSYNKVVNACLETGAYCMIDMHNFARYNGGIIGQGGVSDDIFVDLWVQIAKYYEDNDKIIFGLMNEP
HDLDIEIWAQTCQKVVTAIRKAGATSQMILLPGTNFASVETYVSTGSAEALGKITNPDGSTDLLYFDVHKYLDINNS
GSHAECTTDNVDAFNDFADWLRQNKRQAIISETGASMEPSCMTAFCAQNKAISENSDVYIGFVGWGAGSFDTSYILT
LTPLGKPGNYTDNKLMNECILDQFTLDEKYRPTPTSISTAAEETATATATSDGDAPSTTKPIFREETASPTPNAVTK
PSPDTSDSSDDDKDSAASMSAQGLTGTVLFTVAALGYMLVAF
BsuGluC CBD, bacillus subtilises (Bacillus subtilis) (SEQ ID NO：16)
MKRSISIFITCLLITLLTMGGMIASPASAAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISSHGLQWYGEYVNKD
SLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVIIDWHILNDGNPNQNKEKAKEFFKEMSS
LYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKNDPDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFY
AGTHGQFLRDKANYALSKGAPIFVTEWGTSDASGNGGVFLDQSREWLKYLDSKTISWVNWNLSDKQESSSALKPGAS
KTGGWRLSDLSASGTFVRENILGTKDSTKDIPETPSKDKPTQENGISVQYRAGDGSMNSNQIRPQLQIKNNGNTTVD
LKDVTARYWYKAKN KGQNFDCDYAQIGCGNVTHKFVTLHKPKQGADTYLELGFKNGTLAPGASTGNIQLRLHNDDW
SNYAQSGDYSFFKSNTFKTTKKITLYDQGKLIWGTEPN
BsuXyn3, bacillus subtilises (Bacillus subtilis) xylanase variant (SEQ ID NO：17)
ASTDYWQNWTFGGGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLY
GWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDDTTFTQYWSVRQSKRPTGSNATITF
SNHVNAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW
BsuXyn4, bacillus subtilises (Bacillus subtilis) xylanase variant (SEQ ID NO：18)
ASTDYWQNWTDGYGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLY
GWTRSPLIEYYVVDSWGTYRPTGTYKGTVYSDGGWYDIYTATRDNAPSIDGDFTTFTQYWSVRQSKRPTGSNATITF
SNHVNAWRSHGMDLGSNWAYQVMATEGYLSSGSSNVTVW
Claims (14)
1. the purposes in abnormal flavour in reducing production for the xylanase, wherein said product is food, feedstuff or beverage；Its
Described in xylanase to solubility araboxylan substrate (WE-AX), there is selectivity.
2. the purposes in abnormal flavour in reducing production for the xylanase, wherein said product is brewed beverages.
3. the purposes in abnormal flavour in reducing product preparation process for the xylanase, wherein said process is brewing process.
4. the purposes of xylanase as claimed in one of claims 1-3, wherein said beverage is Fructus Hordei Germinatus beverage.
5. the purposes of xylanase as claimed in one of claims 1-4, wherein said beverage is alcoholic beverage.
6. the purposes of xylanase as claimed in one of claims 1-5, wherein said beverage is medicated beer.
7. the purposes of xylanase as claimed in one of claims 1-6, wherein said purposes includes using described xylanase
Process the step brewageed with converted mash.
8. the purposes of xylanase as claimed in one of claims 1-7, wherein said xylanase is combined with other enzyme
Use.
9. the purposes of xylanase as claimed in one of claims 1-8, the minimizing of wherein said abnormal flavour is the potential of abnormal flavour
Probability and/or the minimizing of abnormal flavour formation.
10. the purposes of xylanase as claimed in one of claims 1-9, the minimizing of wherein said abnormal flavour is that abnormal flavour is formed
Potential feasibility reduces.
The purposes of 11. xylanase as claimed in one of claims 1-10, wherein said abnormal flavour and araboxylan divide
Decorrelation.
12. according to the purposes of the xylanase of any one of claim 1-11, and wherein said abnormal flavour is ferulic acid.
13. according to the purposes of the xylanase of any one of claim 1-12, wherein said xylanase can show inscribe-
1,4- beta-xylanase activity.
14. according to the purposes of the xylanase of any one of claim 1-13, and wherein said xylanase is display inscribe -1,
The enzyme of 4- beta-xylanase activity, this enzyme comprises and selected from SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID
NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:17 and SEQ ID NO:18 any one or they any
Function fragment has the aminoacid sequence of at least 80% homogeneity.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161534574P | 2011-09-14 | 2011-09-14 | |
| US61/534574 | 2011-09-14 | ||
| EP11181241 | 2011-09-14 | ||
| EP11181241.8 | 2011-09-14 | ||
| US201261676535P | 2012-07-27 | 2012-07-27 | |
| US61/676535 | 2012-07-27 | ||
| CN201280044661.3A CN103814129A (en) | 2011-09-14 | 2012-09-14 | Compositions comprising enzymes with endo-1,4-beta-xylanase activity and enzymes with endo-1,3(4)-beta-glucanase activity |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201280044661.3A Division CN103814129A (en) | 2011-09-14 | 2012-09-14 | Compositions comprising enzymes with endo-1,4-beta-xylanase activity and enzymes with endo-1,3(4)-beta-glucanase activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106434246A true CN106434246A (en) | 2017-02-22 |
Family
ID=47883847
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201280044661.3A Pending CN103814129A (en) | 2011-09-14 | 2012-09-14 | Compositions comprising enzymes with endo-1,4-beta-xylanase activity and enzymes with endo-1,3(4)-beta-glucanase activity |
| CN201610802626.3A Pending CN106434246A (en) | 2011-09-14 | 2012-09-14 | Use of xylanase |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201280044661.3A Pending CN103814129A (en) | 2011-09-14 | 2012-09-14 | Compositions comprising enzymes with endo-1,4-beta-xylanase activity and enzymes with endo-1,3(4)-beta-glucanase activity |
Country Status (16)
| Country | Link |
|---|---|
| US (2) | US9683224B2 (en) |
| EP (2) | EP3061816A1 (en) |
| JP (1) | JP6084617B2 (en) |
| CN (2) | CN103814129A (en) |
| AR (1) | AR087885A1 (en) |
| AU (1) | AU2012307299C1 (en) |
| BR (1) | BR112014005873B1 (en) |
| DK (1) | DK2756078T3 (en) |
| EA (2) | EA201790153A3 (en) |
| ES (1) | ES2645921T3 (en) |
| HU (1) | HUE037237T2 (en) |
| MX (1) | MX353745B (en) |
| PL (1) | PL2756078T3 (en) |
| UA (1) | UA128152C2 (en) |
| WO (1) | WO2013037933A2 (en) |
| ZA (1) | ZA201400889B (en) |
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| CN110714037A (en) * | 2019-11-29 | 2020-01-21 | 江南大学 | Preparation method of xylanase and application of xylanase in beer production |
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| CN110295155A (en) * | 2015-12-23 | 2019-10-01 | 东莞泛亚太生物科技有限公司 | Promote durothermic cellulase |
| DK3417057T3 (en) * | 2016-02-18 | 2022-07-04 | Biopract Gmbh | Arabinanase and uses thereof |
| WO2017152169A1 (en) | 2016-03-04 | 2017-09-08 | Danisco Us Inc. | Engineered ribosomal promoters for protein production in microorganisms |
| WO2018049342A1 (en) * | 2016-09-09 | 2018-03-15 | Alpha Revolution, Inc. | Systems, devices, and methods for fermenting beverages |
| WO2019089898A1 (en) | 2017-11-02 | 2019-05-09 | Danisco Us Inc | Freezing point depressed solid matrix compositions for melt granulation of enzymes |
| CN116286751B (en) * | 2023-05-15 | 2023-07-25 | 北京市科学技术研究院 | Bifunctional cellulase mutant with improved catalytic efficiency and application thereof |
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| EA201790153A2 (en) | 2017-06-30 |
| AU2012307299C1 (en) | 2018-10-25 |
| BR112014005873B1 (en) | 2021-11-16 |
| AR087885A1 (en) | 2014-04-23 |
| US20170314004A1 (en) | 2017-11-02 |
| EP3061816A1 (en) | 2016-08-31 |
| HUE037237T2 (en) | 2018-08-28 |
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| UA128152C2 (en) | 2024-04-24 |
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